Method for determination and quantification of radiation or genotoxin exposure

ABSTRACT

The present invention discloses methods for detecting exposure of a living subject to genotoxic agents, testing sensitivity to a genotoxic agent, and determining DNA damage caused by exposure to an agent, comprising detecting the presence of FANCD2-containing foci from a sample collected from said subject. The presence of concentrated foci is indicative of DNA damage, and the degree of foci formation is correlated with degree of exposure. Diagnostic reagents contain a ligand that binds to human FANCD2 associated with a detectable label. Kits for detecting DNA damage in a biological sample contain such diagnostic reagents and signal detection components. The invention further discloses methods for identifying agents which modulate the ability of FANCD2-containing foci to form. Among other things, such agents are potentially useful chemosensitizing agents or may confer protection against damage caused by genotoxic agents.

FIELD OF THE INVENTION

This invention relates to detecting exposure of a living subject togenotoxic agents, such as radiation and environmental toxins. Methodsare also provided which facilitate the identification of therapeuticcompounds for cancer treatment and protection against genotoxic agents.

BACKGROUND OF THE INVENTION

Chemotherapy and radiotherapy constitute common treatments for manydiseases. Although such methods of therapy can be used effectively inthe therapy of diseases such as cancer, exposure to biologicallysignificant levels of radiation can also cause genotoxic stress.Similarly, many industrial processes (such as the production of nuclearpower) and military uses (such as nuclear weapons) can exposeindividuals to hazardous levels of genotoxic agents. Such exposure canelicit a variety of cellular responses, ranging from cell-cycle arrestto mutation, malignant transformation, or cell death.

Due to individual genetic make-up, some people have defective DNA repairmechanisms, resulting in chromosome instability. There are three mainconsequences of such chromosome instability. These individuals may have(1) developmental abnormalities (birth defects), (2) predisposition tocancers, and (3) hypersensitivity of their tissues to radiation andchemotherapy.

In severe cases, such individuals may be born with an obvious geneticdisease (i.e., Fanconi Anemia). This disease results from a completeknockout of both alleles of a particular DNA repair gene. In less severeand more common cases, individuals may have a partial disruption of aDNA repair gene or pathway. Such a disruption may result from inheritinga “variant” DNA repair gene, such as a single nucleotide polymorphism ina Fanconi Anemia (FA) gene. While these individuals may have normaldevelopment (i.e., no evidence of birth defects), the only clinicalsequelae of their genetic weakness may be the early onset of cancer, orradiation or drug sensitivity. The identification of such individuals,before they develop cancer or before they develop life-threateningtoxicity from radiation/drug exposure, is an important, unsolved problemin clinical medicine.

Predicting radiation/chemotherapy toxicity is difficult and relies, atpresent, on circumstantial evidence. First, individuals who have earlyonset of rare cancers or strong family histories of cancer, with clearautosomal dominant or recessive inheritance, may have an underlyinggenetic defect. In these cases, culprit cancer susceptibility genes(BRCA1, BRCA2, p53) can be sequenced to confirm or rule out the defect.Second, individuals who have subtle clinical findings, reminiscent of amore preformed genetic disease (i.e. skin café au lait spots or shortstature), may have an underlying DNA repair disorder.

Efforts to predict which cancer patients have an underlying DNA repairdisorder have been largely ineffectual. While FA patients and Bloomssyndrome patients have obvious (measurable) defects in chromosomebreakage, patients with more subtle DNA repair disorders do not scorepositive in typical chromosome breakage studies. Standard doses ofradiation/chemotherapy are given to all cancer patients, depending onthe specific tumor type and location. Approximately 2% of these patientsmay have unexpected severe toxic reactions, as the first evidence oftheir underlying DNA repair disorder.

There is a need in the art for a method of detecting exposure to agenotoxic agent in a live sample (i.e., a so-called biologicaldosimeter). There is also a need in the art for methods of testing anindividual's sensitivity to a genotoxic agent. There is also a need inthe art for a method of determining damage caused to an individual byexposure to a genotoxic agent. There also exists a need in the art for amethod to identify agents which are active in modulating the responsesof cells toward genotoxic agents. There also exists a considerable needto obtain compounds which are active in protecting an individual fromprobable exposure to a genotoxic agent such as radiation and genotoxiccarcinogens.

SUMMARY OF THE INVENTION

The present invention discloses methods and compositions that are usefulin detecting exposure of a living subject to genotoxic agents. Thesemethods and compositions are based on the observation that FANCD2 andother proteins form nuclear foci in cells exposed to genotoxic agents.The invention also encompasses compositions and methods that are usefulin identifying modulators of foci formation.

In one aspect, the invention provides for a method of detecting exposureto a genotoxic agent in a live subject, comprising the following steps:collecting a sample from the subject; and detecting the presence ofFANCD2-containing foci in the sample. Presence of foci is indicative ofexposure to a genotoxic agent. The subject can be human. The sample canbe selected from a group consisting of peripheral blood, saliva, urine,a cell scraping, exudate, a buccal sample, sputum, and cervicalscraping. In a preferred embodiment, the sample is peripheral blood.Alternatively, the method can further comprise a control sample, wherethe degree of foci formation in the sample relative to a control sampleis indicative of the degree of exposure of the subject to a genotoxicagent.

In one embodiment, the method further comprises contacting a sample witha ligand which binds to human FANCD2 of SEQ ID NO:1. The ligand isassociated with a label which provides a detectable signal. In oneembodiment, the ligand is an antibody. The detectable label can beattached to the antibody. Alternatively, the label is attached to asecond ligand, which binds to the first ligand. The second ligand can bean antibody. The detectable label can be selected from the groupconsisting of colorimetric, chemiluminescent, fluorescent,electrochemical labels and combinations thereof. In a preferredembodiment, the label is a fluorescent dye. In one embodiment, detectioncomprises fluorescence microscopy.

The invention also features a method of testing a patient's sensitivityto a genotoxic agent, comprising the following steps: exposing a patientto a low dose of genotoxic agent; and, detecting the presence ofFANCD2-containing foci relative to a control sample. Presence of fociformation relative to a control sample is indicative of a difference insensitivity of the patient to genotoxic agent. The degree of fociformation relative to a control sample is indicative of the sensitivityof the patient to genotoxic agent. The sample can be selected from agroup consisting of peripheral blood, saliva, urine, a cell scraping,exudate, a buccal sample, sputum, and cervical scraping.

In one embodiment, the method further comprises contacting a sample witha ligand which binds to human FANCD2 of SEQ ID NO:1. The ligand isassociated with a label which provides a detectable signal. In oneembodiment, the ligand is an antibody. The detectable label can beattached to the antibody. Alternatively, the label is attached to asecond ligand, which binds to the first ligand. The second ligand can bean antibody. The detectable label can be selected from the groupconsisting of colorimetric, chemiluminescent, fluorescent,electrochemical labels and combinations thereof. In a preferredembodiment, the label is a fluorescent dye. In one embodiment, detectioncomprises fluorescence microscopy.

In another aspect, the invention provides for a method of determiningthe level of DNA damage caused by exposure of subject to a genotoxicagent, comprising the following steps: collecting a sample from thepatient following the exposure; and detecting the presence ofFANCD2-containing foci relative to a control sample. A difference infoci formation relative to the control sample is indicative of adifference in DNA damage in response to the exposure, and the degree offoci formation relative to a control sample is indicative of a differentextent of DNA damage in response to the exposure. The sample can beselected from a group consisting of peripheral blood, saliva, urine, acell scraping, exudate, a buccal sample, sputum, and cervical scraping.

In another aspect, the invention provides for a method of determiningthe sensitivity of a sample towards genotoxic agents, comprising thefollowing steps: collecting a sample from the patient, exposing thesample or tissue to a genotoxic agent, and detecting the presence ofFANCD2-containing foci relative to a control sample. A difference infoci formation relative to the control sample is indicative of adifference in DNA damage in response to the exposure, and the degree offoci formation relative to a control sample is indicative of a differentsensitivity of the tissue towards the agent. The sample can be tumorsample collected from a living subject. In one embodiment, the genotoxicagent can be selected from the group consisting of ionizing radiation orcisplatin. In another embodiment, samples are taken from the livingsubject at different times to monitor the change in the sensitivity ofthe sample towards the genotoxic agent.

In one embodiment, the method further comprises contacting a sample witha ligand which binds to human FANCD2 of SEQ ID NO:1. The ligand isassociated with a label which provides a detectable signal. In oneembodiment, the ligand is an antibody. The detectable label can beattached to the antibody. Alternatively, the label is attached to asecond ligand, which binds to the first ligand. The second ligand can bean antibody. The detectable label can be selected from the groupconsisting of calorimetric, chemiluminescent, fluorescent,electrochemical labels and combinations thereof. In a preferredembodiment, the label is a fluorescent dye. In one embodiment, detectioncomprises fluorescence microscopy.

In another aspect of the invention, an isolated polynucleotide isprovided which comprises a DNA sequence encoding the human FACD2 proteinof SEQ ID NO:1 fused in frame with a DNA sequence encoding a fluorescentprotein. The polynucleotide can further comprise an expression controlsequence operatively linked to the sequence encoding the FANCD2 proteinfused in frame with a DNA sequence encoding a fluorescent protein. Thefluorescent protein can be selected from the group including: GFP, YFP,CFP, eGFP, eYFP, eCFP, RFP.

In another aspect, the invention provides the protein encoded by anisolated polynucleotide which comprises a DNA sequence encoding thehuman FACD2 protein of SEQ ID NO:1 fused in frame with a DNA sequenceencoding a fluorescent protein. The polynucleotide can further comprisean expression control sequence operatively linked to the sequenceencoding the FANCD2 protein fused in frame with a DNA sequence encodinga fluorescent protein. The fluorescent protein can be selected from thegroup including: GFP, YFP, CFP, eGFP, eYFP, eCFP, RFP.

In yet another aspect, the invention provides a cell expressing anisolated polynucleotide which comprises a DNA sequence encoding thehuman FACD2 protein of SEQ ID NO:1 fused in frame with a DNA sequenceencoding a fluorescent protein. The polynucleotide can further comprisean expression control sequence operatively linked to the sequenceencoding the FANCD2 protein fused in frame with a DNA sequence encodinga fluorescent protein. The fluorescent protein can be selected from thegroup including: GFP, YFP, CFP, eGFP, eYFP, eCFP, RFP.

In another aspect, the invention provides an isolated polynucleotidecomprising a DNA sequence encoding a protein that binds with FANCD2 uponfoci formation, fused in frame with a DNA sequence encoding a secondfluorophore. The nucleic acid encoding a protein that binds with FANCD2upon foci formation can be selected from the group including: Histone2AX, BRCA1, and NBS1. The polynucleotide can further comprise anexpression control sequence operatively linked to the sequence encodinga protein that binds with FANCD2 upon foci formation, fused in framewith a DNA sequence encoding a second fluorophore. The fluorescentprotein can be selected from the group including: GFP, YFP, CFP, eGFP,eYFP, eCFP, RFP. The polynucleotide may further comprise sequenceencoding a protein linker sequence.

In yet another embodiment, the invention provides a protein encoded bythe isolated polynucleotide comprising a DNA sequence encoding a proteinthat binds with FANCD2 upon foci formation, fused in frame with a DNAsequence encoding a second fluorophore. The nucleic acid encoding aprotein that binds with FANCD2 upon foci formation can be selected fromthe group including: Histone 2AX, BRCA1, and NBS1. The polynucleotidecan further comprise an expression control sequence operatively linkedto the sequence encoding a protein that binds with FANCD2 upon fociformation, fused in frame with a DNA sequence encoding a secondfluorophore. The fluorescent protein can be selected from the groupincluding: GFP, YFP, CFP, eGFP, eYFP, eCFP, RFP. The polynucleotide mayfurther comprise sequence encoding a protein linker sequence.

In yet another embodiment, the invention provides a cell expressing theisolated polynucleotide comprising a DNA sequence encoding a proteinthat binds with FANCD2 upon foci formation, fused in frame with a DNAsequence encoding a second fluorophore. The nucleic acid encoding aprotein that binds with FANCD2 upon foci formation can be selected fromthe group including: Histone 2AX, BRCA1, and NBS1. The polynucleotidecan further comprise an expression control sequence operatively linkedto the sequence encoding a protein that binds with FANCD2 upon fociformation, fused in frame with a DNA sequence encoding a secondfluorophore. The fluorescent protein can be selected from the groupincluding: GFP, YFP, CFP, eGFP, eYFP, eCFP, RFP. The polynucleotide mayfurther comprise sequence encoding a protein linker sequence.

In one embodiment, the invention provides a cell expressing the isolatedpolynucleotide a DNA sequence encoding the human FACD2 protein of SEQ IDNO:1 fused in frame with a DNA sequence encoding a first fluorescentprotein and isolated polynucleotide comprising a DNA sequence encoding aprotein that binds with FANCD2 upon foci formation, fused in frame witha DNA sequence encoding a second fluorescent protein. The secondfluorescent protein is preferably different from the first fluorescentprotein. The polynucleotides may further comprise sequences encoding aprotein linker sequences. The polynucleotides may further compriseexpression control sequences operatively linked to the sequence encodingthe proteins fused in frame with the DNA sequence encoding a fluorescentprotein. The fluorescent proteins can be selected from the groupincluding: GFP, YFP, CFP, eGFP, eYFP, eCFP, RFP. In a preferredembodiment, the fluorescent proteins are eCFP and eYFP.

In yet another aspect, the invention provides a method of screening testagents which modulate formation of FANCD2-containing foci, comprisingthe following steps: contacting a biological sample with test compound;and detecting the presence of FANCD2 in FANCD2-containing foci relativeto a control sample. A difference in the degree of foci formationrelative to a control sample is indicative of an agent active in fociformation. The method may further comprise exposing biological sample toa genotoxic agent.

In one embodiment, the method further comprises contacting thebiological sample with a ligand which binds to FANCD2. The ligand isassociated with a label which provides a detectable signal. In oneembodiment, the ligand is an antibody. The detectable label can beattached to the antibody. Alternatively, the label is attached to asecond ligand, which binds to the first ligand. The second ligand can bean antibody. The detectable label can be selected from the groupconsisting of calorimetric, chemiluminescent, fluorescent,electrochemical labels and combinations thereof. In a preferredembodiment, the label is a fluorescent dye. In one embodiment, detectioncomprises fluorescence microscopy.

In another embodiment, the method further comprises detecting fociformation using a cell expressing an isolated polynucleotide whichcomprises a DNA sequence encoding the human FACD2 protein of SEQ ID NO:1fused in frame with a DNA sequence encoding a fluorescent protein. Thepolynucleotide can further comprise an expression control sequenceoperatively linked to the sequence encoding the FANCD2 protein fused inframe with a DNA sequence encoding a fluorescent protein. Thefluorescent protein can be selected from the group including: GFP, YFP,CFP, eGFP, eYFP, eCFP, RFP. In one embodiment, foci are detected usingfluorescence microscopy.

In yet another embodiment, the method further comprises detecting fociformation using a cell expressing the isolated polynucleotide a DNAsequence encoding the human FACD2 protein of SEQ ID NO:1 fused in framewith a DNA sequence encoding a first fluorescent protein and isolatedpolynucleotide comprising a DNA sequence encoding a protein that bindswith FANCD2 upon foci formation, fused in frame with a DNA sequenceencoding a second fluorescent protein. The second fluorescent protein ispreferably different from the first fluorescent protein. Thepolynucleotides may further comprise sequences encoding a protein linkersequences. The polynucleotides may further comprise expression controlsequences operatively linked to the sequence encoding the proteins fusedin frame with the DNA sequence encoding a fluorescent protein. Thefluorescent proteins can be selected from the group including: GFP, YFP,CFP, eGFP, eYFP, eCFP, RFP. In a preferred embodiment, the fluorescentproteins are eCFP and eYFP. In another embodiment, foci are detected bemeasuring fluorescence resonance energy transfer.

In another aspect, the invention provides a method of protecting aliving subject from damage caused by genotoxic agents, the methodcomprising administering a therapeutically effective amount of anenhancer of FANCD2-containing foci formation to a living subject, suchthat formation of FANCD2-containing foci formation is enhanced in theliving subject when compared to a reference or control. In oneembodiment, the enhancer comprises desferrioxamine.

In yet another aspect, the invention provides an antibody that binds toa monoubiquitinated form of FANCD2 polypeptide. Preferably, the antibodybinds the monoubiquitinated and not the unubiquitinated form of FANCD2polypeptide. The antibody may be polyclonal. Alternatively, the antibodymay be monoclonal.

In another aspect, the invention provides for a kit for detecting thepresence or absence of FANCD2-containing foci in a sample from a livesubject comprising the antibody that binds to a monoubiquitinated formof FANCD2, and packaging material. The kit may further comprise afluorescently labeled secondary antibody, wherein the secondary antibodybinds to the first antibody.

Other aspects and advantages of the present invention are describedfurther in the following detailed description of the preferredembodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic model of the GFP (Green Fluorescent Protein) fusion toFANCD2. A cDNA, encoding GFP-fused directly to the amino terminalmethionine (MET) of FANCD2, was generated. Transfection of mammaliancells with this cDNA results in expression of the fusion protein, whichfunctions in a reporter assay.

FIG. 2. The GFP-FANCD2 fusion protein is expressed and activated bymonoubiquitination in transfected FA-D2 human fibroblasts.FANCD2-deficient human fibroblasts (PD20F) were either untransfected(lane 1), transfected with wild-type FANCD2 cDNA (lane 2-5), ortransfected with the cDNA encoding the GFP-FANCD2 fusion protein (lane6-9), as indicated. Alternatively, FA-A fibroblasts (GM6914, lanes11-14) or HeLa cells (lanes 16-19) were transfected with the cDNAencoding the fusion protein. Cells were either untreated or exposed togenotoxic stress (ionizing radiation), as indicated. Whole cell lysateswere analyzed by SDS-PAGE, and cellular proteins were immunoblotted withantisera to FANCD2. Conclusion: DNA damage activates the increasedmonoubiquitination of either the full-length (wild-type) FANCD2 proteinor the GFP-FANCD2 fusion protein in corrected cells but not in FA cells.

FIG. 3. The GFP-FANCD2 fusion protein functionally complements theMitomycin C hypersensitivity of FANCD2-deficient human fibroblasts. Thetransfected PD20F cells, stably expressing GFP-FANCD2 protein, wereanalyzed for survival in variable concentrations of MMC. Conclusion:GFP-FANCD2 is functional, and it corrects the MMC hypersensitivity ofPD20F cells. The GFP moiety at the amino terminus therefore does notinterfere with its function in the cell.

FIG. 4. Isolation of a PD20F fibroblast clone, expressing a physiologiclevel of the GFP-FANCD2 protein and suitable for high throughput drugscreening. A subclone (clone 7) was isolated, by limiting dilution,which expresses GFP-FANCD2 protein. Activation of this clone withionizing radiation (IR) results in the assembly of bright green foci,reading detectable in the fluorescence background of the cell nucleus.

FIG. 5. A general method for screening for inhibitors and agonists ofthe Fanconi Anemia/BRCA pathway. Human fibroblasts (clone 7), stablyexpressing GFP-FANCD2, are exposed, in 384 well plates, to potentialsmall molecule inhibitors or agonists compounds. Following pretreatmentwith these compounds, the plates are irradiated, to activate themonoubiquitination and foci formation of GFP-FANCD2. GFP-FANCD2 fails toform IR-inducible foci when expressed stably in a FA-A fibroblast. Asmall molecule agonist, which bypasses the FA enzyme complex, activatesthe foci formation in FA cells. Such a drug may be a suitable treatmentfor FA. Therefore, the GFP-FANCD2 fusion protein is used to screeneither for inhibitors or agonists of the pathway.

FIG. 6. Schematic protocol for the identification of small moleculeinhibitors and agonists of the FA/BRCA pathway. Human fibroblasts,stably expressing GFP-FANCD2, are plated in 384-well plates, pretreatedwith chemical libraries, and exposed to Ionizing Radiation. GFP foci arescored, leading to the identification of inhibitors or agonists of thepathway.

FIG. 7. Identification of Wortmannin and Trichostatin-A as inhibitors ofthe FA/BRCA pathway. Using the strategy outlined in FIG. 6, a chemicallibrary, containing over 1000 independent compounds was screened. Mostcompounds had no effect on the formation of IR-inducible GFP-FANCD2foci. Two compounds (Trichostatin A, a known HDAC inhibitor andwortmannin, a known ATR-kinase inhibitor) efficiently blocked fociformation. Taken together, these results suggest that cellular HDACactivity (i.e., histone deacetylase activity) and ATR-kinase activityare required upstream in the FA/BRCA pathway. The ability ofTrichostatin-A and Wortmannin to inhibit foci formation was confirmed indose-response and time course studies (date not shown) subsequently. Theactivity of these agents in this in vitro assay suggests that theseagents will chemosensitize tumors to radiation and chemotherapy in vivo.

FIG. 8. The cellular signaling proteins ATR, RPA1, and CHK1 are requiredupstream in the FA/BRCA signaling pathway. The inhibition of GFP-FANCD2foci assembly by wortmannin suggested that the RPA1/ATR/CHK1 pathway mayfunction as a sensor upstream in this pathway. siRNA inhibition was usedto block the RPA1/ATR/CHK1 pathway. Transient transfection with siRNAsfor RPA1, ATR, or CHK1 resulted in loss of expression of thecorresponding cellular proteins. Interestingly, there was a decrease inthe MMC (A) and IR (B) inducible monoubiquitination of FANCD2 in thesetreated cells, as judged by the decrease in FANCD2-L/FANCD2-S ratio.Cells were transfected with siRNA specific for the FANCA gene (lanes 9,10). siRNA to FANCA decreased FANCA expression (not shown) and decreasedthe FANCD2-L/FANCD2-S ratio, further demonstrating that FANCA worksupstream in the FA pathway.

FIG. 9. Schematic model of the FA/BRCA pathway, showing upstreamRPA1/ATR/CHK1 sensor apparatus. The general approach of siRNA inhibitionis used to identify other upstream proteins in the FA/BRCA pathway.Inhibition of any of these upstream targets (say, inhibition of ATR, byWortmannin) is a potential mechanism of knocking out the pathway andsensitizing human tumor cells to cisplatin or IR.

FIG. 10. Generation of a monoclonal antibody specific for themonoubiquitinated (activated) isoform of FANCD2. A thirteen amino acidpeptide, corresponding to an internal region of human FANCD2 (aminoacids 554-566) was generated. This peptide contains lysine 561, the siteof monoubiquitination. Via a gamma peptide linkage, we coupled a sevenamino acid peptide (GGRLRLC) (SEQ ID NO:3), corresponding to thecarboxyl terminus of ubiquitin to K561. This antigen was then coupled toKLH and used to immunize mice. Mice which developed a serologic responseto the coupled antigen were sacrificed, and splenocyte fusion, formonoclonal antibody production, were performed.

FIG. 11. Generation of hybridomas expressing monoclonal antibodies,specific for monoubiquitinated FANCD2. Following splenocyte fusion,murine hybridomas were subcloned, and supernatants were analyzed fortheir ability to recognize monoubiquitinated FANCD2 by western blotanalysis. Six hybridomas were identified which specifically recognizedmonoubiquitinated FANCD2 by immunoblot, but did not recognize unmodifiedFANCD2. An antibody to monoubiquitinated FANCD2 provides a rapid FACS(fluorescent activated cell sorter) screen for peripheral bloodlymphocytes (PBLs) which have been activated by radiation exposure.

FIG. 12. Dose-dependent Generation of FANCD2 Monoubiquitination afterIR. Exponentially growing HeLa cells were either untreated or exposed tothe indicated IR dose. After four hours, cells were processed byanti-FANCD2 western blotting. Note that some activation of FANCD2monoubiquitination can be observed in the low IR dose range (0-5 Gyradiation range).

FIG. 13. Dose-dependent and Time-Course Dependent Generation of FANCD2Monoubiquitination and FANCD2 Foci formation after Genotoxic Stress.Exponentially growing HeLa cells were either untreated or exposed to theindicated genotoxic agents (Mitomycin C, gamma irradiation, orultraviolet light) and processed by western blotting (panels A, B, C) orimmunofluorescence (panel D) with the polyclonal anti-FANCD2 antibody(E35).

DETAILED DESCRIPTION

The invention is based upon the observation that FANCD2-containing fociare formed in cells in response to exposure to genotoxic agents.Detection of FANCD2-containing foci in samples from living subjects,therefore, may be used to measure exposure of the subject to suchgenotoxic agents.

In order to more clearly and concisely describe and point out thesubject matter of the claimed invention, the following definitions areprovided for specific terms which are used in the following writtendescription and the appended claims.

Definitions

A “genotoxic agent” or “genotoxin” refers to any chemical compound ortreatment method that induces DNA damage when applied to a cell. Suchagents can be chemical or radioactive. A genotoxic agent is one forwhich a primary biological activity of the chemical (or a metabolite) isalteration of the information encoded in the DNA. Genotoxic agents canvary in their mechanism of action, and can include: alkylating agentssuch as ethylmethane sulfonate (EMS), nitrosoguanine and vinyl chloride;bulky addition products such as benzo(a)pyrene and aflatoxin B1;reactive oxygen species such as superoxide, hydroxyl radical; baseanalogs such as 5-bromouracil; intercalating agents such as acridineorange and ethidium bromide. A variety of chemical compounds, alsodescribed as “chemotherapeutic agents,” function to induce DNA damage.Chemotherapeutic agents contemplated to be of use include, e.g.,adriamycin, 5-fluorouracil (5FU), etoposide (VP-16), camptothecin,actinomycin-D, mitomycin C, cisplatin (CDDP) and even hydrogen peroxide.“Genotoxic agents” also include radiation and waves that induce DNAdamage such as γ-irradiation, X-rays, UV-irradiation, microwaves,electronic emissions, and the like. In addition, certain chemicals,sometimes called indirect genotoxic agents, can be converted intogenotoxic agents by normal metabolic enzymes. As used herein, genotoxicagents refer to both direct and indirect genotoxic agents. Genotoxicagents cause mutations in DNA, and can cause cancer. The term “genotoxicagents” also encompasses the use of a combination of one or more DNAdamaging agents, whether radiation-based or actual compounds.

Because of the wide diversity of genotoxic agents, exposure to genotoxicagents comes in many different forms. Mechanism of exposure to chemicalgenotoxic agents may include direct contact, or inhalation by thesubject. In the case of radiation, exposure may arise from proximity toa source of ionizing radiation. The nature of exposure to thesegenotoxic agents can also vary. Exposure can be deliberate, as is thecase with chemotherapy and radiotherapy, but may also be accidental.Examples of accidental exposure may include occupational chemicalexposure in a laboratory, factory or farm, or occupational exposure toionizing radiation in a nuclear power plant, clinic, laboratory, or byfrequent airplane travel.

“DNA damage”, as used herein, refers to chemical and/or physicalmodification of the DNA in a cell, including methylation, alkylationdouble-stranded breaks, cross-linking, thymidine dimers caused byultraviolet light, and oxidative lesions formed by oxygen radicalbinding to DNA bases.

A “ligand” of a protein includes a substrate and other compounds thatbind to the protein. In one embodiment, a “ligand” is an antibody thatbinds to FANCD2. The term “antibody to FANCD2”, or “antibody that bindsto FANCD2”, as used herein, refers to an immunoglobulin molecule whichis able to specifically bind to FANCD2. Antibodies can be intactimmunoglobulins derived from natural sources or from recombinantsources, or immunoreactive portions of intact immunoglobulins.Antibodies are typically tetramers of immunoglobulin molecules. Theantibodies in the present invention exist in a variety of formsincluding, for example, high affinity polyclonal antibodies, monoclonalantibodies, synthetic antibodies, chimeric antibodies, recombinantantibodies and humanized antibodies. Such antibodies originate fromimmunoglobulin classes IgG, IgM, IgA, IgD and IgE. The term antibodyalso includes synthetic antibodies which are generated using recombinantDNA technology, such as, for example, an antibody expressed by abacteriophage. In one embodiment, a desirable ligand is a monoclonalantibody which binds to monoubiquitinated FANCD2, for example asdescribed in detail in Example 1. Other such antibodies include a Fab,Fab′ or F(ab′)2, or Fe antibody fragment thereof which binds FANCD2. Theinvention also provides for a ligand that is a single chain Fv antibodyfragment which binds FANCD2.

Another useful “ligand” is a recombinant construct comprising acomplementarity determining region of an antibody, a synthetic antibodyor a chimeric antibody construct which shares sufficient CDRs to retainthe functionally equivalent binding characteristics of an antibody thatbinds FANCD2.

The term “antibody” includes an antibody which has been generated by thesynthesis of a DNA molecule encoding the antibody and which DNA moleculeexpresses an antibody protein, or an amino acid sequence specifying theantibody, wherein the DNA or amino acid sequence has been obtained usingsynthetic DNA or amino acid sequence technology which is available andwell known in the art.

As used herein, “immunological methods” means any assay involvingantibody-based detection techniques well-known in the art, including,without limitation, Western blotting, immunoprecipitation, FACSanalysis, immunofluorescence microscopy, immunohistochemistry and directand competitive ELISA and RIA techniques (Harlow et al., 1989,Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Ausubel etal., 1995, Current Protocols in Molecular Biology, John Wiley & Sons,Inc., New York; Sambrook et al., 1989, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press).

As used herein, “subject” refers to an animal including mammal, andincluding human, cow, mouse, rat, pig, and sheep. The subject may be ofany age or developmental stage. Furthermore, the subject may be healthy,diseased, or contain mutations within its genome. The subject may alsohave been previously exposed to a genotoxic agent, a therapeutic agentor a test agent. Furthermore, non-human subjects may additionallyinclude transgenic animals. In particular, the subject may carrytransgenes or mutations which alter the organism's sensitivity togenotoxic agents.

By “sample” is meant any cell or tissue, or cell or tissue-containingcomposition or isolate derived from the subject. The sample may bederived from heart, brain, placenta, liver, skeletal muscle, kidney,pancreas, spleen, thymus, prostate, testis, uterus, small intestine, orcolon. Another type of biological sample may be a preparation containingwhite blood cells, e.g., peripheral blood, sputum, saliva, urine, etc.for use in detecting the presence or absence of DNA damage in a subjectthat has been exposed to a genotoxic agent, such as radiation,chemicals, etc.

As used in the first aspect of the invention, a “control sample” refersto a sample isolated in the same way as the sample to which it iscompared, except that the control sample is not exposed to a genotoxicagent.

As used herein, a “reference sample” refers to a sample from a subjectthat is distinct from the test subject and isolated in the same way asthe sample to which it is compared, and which has been exposed to aknown quantity of the same genotoxic agent. The subject of the referencesample may be genetically identical to the test subject, or may bedifferent. In addition, the reference sample may be derived from severalsubjects who have been exposed to a known quantity of the same genotoxicagent.

By “difference in foci formation” is meant an increase or decrease inthe number, size or persistence of FANCD2-containing foci, whencomparing a test sample with either a control sample or referencesample. A difference includes an increase or decrease that is 2-fold ormore, or less, for example 5, 10, 20, 100, 1000-fold or more as comparedto a control or reference sample. A difference also includes an increaseor decrease that is 5% more or less, for example, 10%, 20%, 30%, 50%,75%, 100%, as compared to a control or reference sample.

As used herein, exposure to a “low level” of a genotoxic refers toexposure to a dose of a particular genotoxic agent which results in nomore than 20% of the maximal number of FANCD2-containing foci inbiological samples. Because of the multitude of genotoxic agents towhich a sample may be exposed, as well as the varying sensitivities ofdifferent samples to such genotoxic agents, it is preferable to expressthe dosage relative to the formation of FANCD2-containing foci, ratherthan in the absolute dose of a particular genotoxic agent.

As used herein, “FANCD2-containing foci” are protein complexescomprising FANCD2 which form within nuclei of cells upon exposure togenotoxic agents such as ionizing radiation, or alkylating reagents.FANCD2-containing foci may also contain several other DNA damageresponse proteins, including Histone 2AX, BRCA1, RAD51, BRCA2, and NBS1.Recent studies suggest that, for ionizing radiation inducible foci, theHistone 2AX protein enters the foci early, followed by BRCA1 and NBS1,followed by FANCD2. The relative speed of foci formation, the durationof foci, and the composition of foci (i.e., which proteins interact withFANCD2) may correlate with different kinds of genotoxic stress (i.e., IRversus alkylating agent versus crosslinker stress).

As used herein, a “protein associated with FANCD2-containing foci”refers to a protein which associates with FANCD2 specifically uponformation of FANCD2-containing foci. These proteins, which include theBRCA1 and NBS1 proteins, show interaction with FANCD2 inFANCD2-containing foci, which form upon DNA damage. At this time, littleis known about the nature of the binding interactions among theseproteins. Many of the proteins may bind indirectly, through intermediateproteins and post-translational modifications.

By “detecting FANCD2-containing foci” is meant detecting FANCD2 withinfoci which contain FANCD2. FANCD2 is monoubiquitinated prior toassociation within foci. Therefore, in addition to immunological methodsincluding immunofluorescence detection of foci using ligands which bindto FANCD2, “detecting FANCD2-containing foci” includes detection ofmonoubiquitinated FANCD2 in an extract from a sample using ligands whichspecifically bind to the monoubiquitinated, but not the unubiquitinated,form of FANCD2 using immunological methods. Finally, detectingFANCD2-containing foci can also comprise detecting FANCD2 withinFANCD2-containing foci through the use of a FANCD2 protein which isassociated with a detectable label, for example, FANCD2 protein fused toa fluorescent protein. FANCD2-containing foci can be detected as earlyas 30 minutes after exposure to genotoxic stress. However, FANCD2 fociappear to reach a maximum between 8 and 24 hours after exposure.

As used herein, “sensitivity” of a subject to a genotoxic agent refersto the response of an individual subject to a defined dose of agenotoxic agent. A subject is considered sensitive to a genotoxic agentwhen the subject's DNA is at least 50% more susceptible to damage, forexample 55%, 60%, 75%, 100% over a specified time period, or 2-fold ormore susceptible to damage, for example, 5-fold, 10-fold, 25-fold,50-fold, 100-fold or 1000-fold more susceptible over a specified timeperiod, compared with a normal subject. DNA damage can be measured interms of the number of mutations, double-stranded breaks, or any othermeans known in the art.

By “degree of foci formation” refers to the total number or the rate offormation of FANCD2-containing foci in a sample. The degree of fociformation can be normalized from one sample to another, for example, tototal number of cells, total number of intact nuclei, total samplevolume, or total sample mass. As used herein, a cell nucleus isconsidered “positive for FANCD2-containing foci” if there are greaterthan five bright foci in the nucleus.

“Modulate” formation of FANCD2-containing foci, as used herein, refersto a change or an alteration in the formation of FANCD2-containing fociin a biological sample. Modulation may be an increase or a decrease infoci number, size or persistence within a biological sample, andincludes an increase or decrease that is 2-fold or more, or less, forexample 5, 10, 20, 100, 1000-fold or more as compared to a control orreference sample. Modulation may also be an increase or decrease that is5% more or less, for example, 10%, 20%, 30%, 50%, 75%, 100%, as comparedto a control or reference sample.

The term “modulator” refers to a chemical compound (naturally occurringor non-naturally occurring), such as a biological macromolecule (e.g.,nucleic acid, protein, non-peptide, or organic molecule), or an extractmade from biological materials such as bacteria, plants, fungi, oranimal (particularly mammalian) cells or tissues, or even an inorganicelement or molecule. Modulators are evaluated for potential activity asinhibitors or activators (directly or indirectly) of a biologicalprocess or processes (e.g., agonist, partial antagonist, partialagonist, antagonist, antineoplastic agents, cytotoxic agents, inhibitorsof neoplastic transformation or cell proliferation, cellproliferation-promoting agents, and the like) by inclusion in screeningassays described herein. The activities (or activity) of a modulator maybe known, unknown or partially-known. Such modulators can be screenedusing the methods described herein.

The term “candidate modulator” refers to a compound to be tested by oneor more screening method(s) of the invention as a putative modulator.Usually, various predetermined concentrations are used for screeningsuch as 0.01 μM, 0.1 μM, 1.0 μM, and 10.0 μM, as described more fullybelow. Test compound controls can include the measurement of a signal inthe absence of the test compound or comparison to a compound known tomodulate the target.

As used herein, an “enhancer” of FANCD2-containing foci formation refersto a chemical compound which causes an increase in the formation ofFANCD2-containing foci in a living subject or a biological sample.Enhancement may be an increase in number, size or persistence ofFANCD2-containing foci, and includes an increase that is 2-fold or more,for example, 2, 5, 10, 20, 100, 1000-fold or more as compared to acontrol or reference. Enhancement may also be an increase of 5% or more,for example 5%, 10%, 20%, 30%, 50%, 75%, 100% or more, as compared to acontrol or reference.

As used herein, an “inhibitor” of FANCD2-containing foci formationrefers to a chemical compound which causes a decrease in the formationof FANCD2-containing foci in a living subject or a biological sample.Inhibition may be a decrease in number, size or persistence ofFANCD2-containing foci, and includes a decrease that is 2-fold or more,for example, 2, 5, 10, 20, 100, 1000-fold or more as compared to acontrol or reference. Inhibition may also be an decrease of 5% or more,for example 5%, 10%, 20%, 30%, 50%, 75%, or up to 100%, as compared to acontrol or reference.

A “fusion protein” is a protein that contains at least two polypeptideregions and, optionally, a linking peptide that operatively link the twopolypeptides into one continuous polypeptide. The at least twopolypeptide regions in a fusion protein are derived from differentsources, and therefore a fusion protein comprises two polypeptideregions not normally joined together in nature. The at least twopolypeptides can be joined in any order.

A “linking sequence (or linker peptide)” contains one or more amino acidresidues joined via peptide bonds. A linking sequence serves to join twopolypeptide regions of differing origins in a fusion protein via apeptide bond between the linking sequence and each of the polypeptideregions. Preferably, a length of linking sequence is between 1 and 20amino acids in length, and more preferably between 3 and 10 amino acidsin length.

Typically, a fusion protein is synthesized as a continuous polypeptidein a recombinant host cell which contains an expression vectorcomprising a nucleotide sequence encoding the fusion protein wherein thedifferent regions of the fusion protein are fused in frame on eitherside of a linker peptide's coding sequence. The chimeric coding sequence(encoding the fusion protein) is operatively linked to expressioncontrol sequences (generally provided by the expression vector) that arefunctional in the recombinant host cell.

The term “vector” or “expression vector” refers to a DNA constructcontaining a DNA sequence which is operably linked to a suitable controlsequence capable of effecting the expression of the DNA in a suitablehost. Such control sequences include a promoter to effect transcription,an optional operator sequence to control such transcription, a sequenceencoding suitable mRNA ribosome binding sites, and sequences whichcontrol termination of transcription and translation. The vector may bea plasmid, a phage particle, or simply a potential genomic insert. Oncetransformed into a suitable host, the vector may replicate and functionindependently of the host genome, or may, in some instances, integrateinto the genome itself. In the present specification, “plasmid” and“vector” are sometimes used interchangeably as the plasmid is the mostcommonly used form of vector at present. However, the invention isintended to include such other forms of expression vectors which serveequivalent functions and which are, or become, known in the art.

“Operatively-linked” refers to polynucleotide sequences which arenecessary to effect the expression of coding and non-coding sequences towhich they are ligated. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence; in eukaryotes, generally, such control sequencesinclude promoters and transcription termination sequence. The term“control sequences” is intended to include, at a minimum, componentswhose presence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences.

As used herein, a “pharmaceutical composition” comprises apharmacologically effective amount of a modulator of FANCD2-containingfoci formation and a pharmaceutically acceptable carrier. As usedherein, “pharmacologically effective amount,” “therapeutically effectiveamount” or simply “effective amount” refers to that amount of amodulator of FANCD2-containing foci formation, effective to produce theintended pharmacological, therapeutic or preventive result. For example,if a given clinical treatment is considered effective when there is atleast a 25% enhancement in foci formation, a therapeutically effectiveamount is the amount necessary to effect at least a 25% reduction inthat parameter.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof. The term specifically excludes cellculture medium. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract.

The term “pharmaceutically acceptable salt” refers to both acid additionsalts and base addition salts. The nature of the salt is not critical,provided that it is pharmaceutically acceptable. Exemplary acid additionsalts include, without limitation, hydrochloric, hydrobromic,hydroiodic, nitric, carbonic, sulphuric, phosphoric, formic, acetic,citric, tartaric, succinic, oxalic, malic, glutamic, propionic,glycolic, gluconic, maleic, embonic (pamoic), methanesulfonic,ethanesulfonic, 2-hydroxyethanesulfonic, pantothenic, benzenesulfonic,toluenesulfonic, sulfanilic, mesylic, cyclohexylaminosulfonic, stearic,algenic, β-hydroxybutyric, malonic, galactaric, galacturonic acid andthe like. Suitable pharmaceutically acceptable base addition saltsinclude, without limitation, metallic salts made from aluminum, calcium,lithium, magnesium, potassium, sodium and zinc or organic salts madefrom N,N′-dibenzylethylenediamine, chloroprocaine, choline,diethanolamine, ethylenediamine, N-methylglucamine, lysine, procaine andthe like. Additional examples of pharmaceutically acceptable salts arelisted in Journal of Pharmaceutical Sciences (1977) 66:2. All of thesesalts may be prepared by conventional means from a modulator ofFANCD2-containing foci by treating the compound with the appropriateacid or base.

FANCD2 Foci

The cellular response to DNA damage is a complex interacting network ofpathways that mediate cell cycle checkpoints, DNA repair, and apoptosis.A model lesion for the investigation of these pathways has been DNAdouble-strand breaks, which rapidly induce cell cycle checkpoints andare repaired by a number of different pathways. In mammalian cells, bothhomologous recombination and nonhomologous recombination pathways areutilized. Extensive studies in mammalian cells have shown that complexesof DNA repair and cell cycle checkpoint proteins rapidly localize tosites of double-strand breaks induced by ionizing radiation. Theseproteins create foci that can be detected by immunofluorescent analyses.

The Fanconi anemia complementation group D2 (FANCD2) is a component of aprotein complex involved in chromosome stability and repair. Fanconianemia (FA) is a hereditary disorder characterized, in part, by adeficient DNA-repair mechanism that increases a person's risk for avariety of cancers. In response to DNA damage, the FA complex activatesFANCD2, which then associates with BRCA1. Activation of FANCD2 occurs byphosphorylation of a serine 222 residue by the ATM kinase. In addition,activation via the FA pathway occurs via monoubiquitination of FANCD2 atlysine 561. In its unmodified form, FANCD2 is diffusely locatedthroughout the nucleus. When ubiquitinated, FANCD2 forms dots, or foci,in the nucleus. The ubiquitination of FANCD2 and subsequent formation ofnuclear foci occurs in response to DNA damage. By coimmunoprecipitation,Nakanishi et al. (2002) found constitutive interaction between FANCD2and NBS1, and they provided evidence that these proteins interact in twodistinct assemblies to mediate S-phase checkpoint and resistance tomitomycin C-induced chromosome damage.

At least two types of ionizing radiation-induced foci have beenobserved: one containing the Rad51, BRCA1 and BRCA2 proteins, andanother containing the Mre11-Rad50-NBS1 complex. Rad51 foci, whichcontain the tumor suppressor proteins BRCA1 and BRCA2, also appearduring S phase in the absence of exogenous induction of DNA damage.

Mre11-Rad50-NBS1 foci can be detected as early as 10 min afterirradiation and are clearly present at sites of DNA breaks, while DNArepair is ongoing. These foci also colocalize with the BRCA1 protein,which has been shown to be required for their formation, possiblythrough its physical interaction with human Rad50 (hRad50). In addition,coimmunoprecipitation experiments performed with BRCA1 have indicatedthe presence of a large number of additional proteins in this complex(referred to as the BRCA1-associated surveillance complex). Theseinclude the mismatch repair proteins Msh2, Msh6, and Mlh1, thecheckpoint kinase ATM, the product of the Bloom's syndrome gene BLM, andreplication factor C. BRCA1, NBS1, and hMre11 have all been shown to besubstrates of the ATM kinase and to become phosphorylated in response tothe presence of DNA breaks.

The present invention is related to the discovery that cells exposed togenotoxic agents form FANCD2-containing foci. Multiple DNA damageresponse proteins have now been identified which form nuclear foci, alsocalled IRIFs (Ionizing-Radiation Inducible foci) in response to DNAdamage.

Dosage- and Time-Dependence of Foci Formation in Response to GenotoxicAgent

In vitro studies show that cellular exposure to Ionizing Radiation (inthe 0.5 to 5 Gy range) results in a dose-dependent increase in FANCD2monoubiquitination and foci formation. Foci can be detected as early as30 minutes after IR exposure, and peak foci are observed between 8 and24 hours after exposure. Foci formation is delayed (compared to this IRresponse) after cellular exposure to toxic drugs, such as cisplatin andmitomycin C (MMC). This delay probably reflects the slow uptake of thesedrugs and the requirement for metabolic activation before the DNA isactually damaged. Importantly, these studies have been done in vitro.

Ligands According to the Invention

Ligands useful for the invention includes antibodies that bind toFANCD2. Antibodies that bind to FANCD2 have been described(US20030093819A1; Garcia-Higuera et al., 2001. Mol. Cell. 7:249-62).Alternatively antibodies that bind to FANCD2 can be generated byconventional means utilizing the isolated, recombinant or modifiedFANCD2 or fragments thereof as antigens of this invention. For example,polyclonal antibodies are generated by conventionally stimulating theimmune system of a selected animal or human with a FANCD2 antigen,allowing the immune system to produce natural antibodies thereto, andcollecting these antibodies from the animal or human's blood or otherbiological fluid. Preferably a recombinant version of FANCD2 is used asan immunogen. Monoclonal antibodies (MAbs) directed against FANCD2 arealso generated conventionally. Hybridoma cell lines expressing desirableMAbs are generated by well-known conventional techniques, e.g. Kohlerand Milstein and the many known modifications thereof. Similarlydesirable high titer antibodies are generated by applying knownrecombinant techniques to the monoclonal or polyclonal. antibodiesdeveloped to these antigens [see, e.g., PCT Patent Application No.PCT/GB85/00392; British Patent Application Publication No. GB218863 8A;Amit et al., 1986 Science, 233:747-753; Queen et al., 1989 Proc. Nat'l.Acad. Sci. USA, 86:10029-10033; PCT Patent Application No.PCT/WO9007861; and Riechmann et al., Nature, 332:323-327 (1988); Huse etal, 1988a Science, 246:1275-128 1].

Given the disclosure contained herein, one of skill in the art generatesligands or antibodies directed against FANCD2 by techniques known in theart, for example, by manipulating the complementarity determiningregions of animals or human antibodies to the antigen of this invention.See, e.g., E. Mark and Padlin, “Humanization of Monoclonal Antibodies”,Chapter 4, The Handbook of Experimental Pharmacology, Vol. 113, ThePharmacology of Monoclonal Antibodies, Springer-Verlag (June, 1994);Harlow et al, 1999, Using Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: ALaboratory Manual, Cold Spring Harbor, N.Y.; Houston et al, 1988, Proc.Natl. Acad. Sci. USA 85:5879-5883; and Bird et al, 1988, Science242:423-426.

Alternatively, FANCD2 antigens are assembled as multi-antigeniccomplexes [see, e.g., European Patent Application 0339695, publishedNov. 2, 1989] and employed to elicit high titer antibodies capable ofbinding the FANCD2. The present invention also provides anti-idiotypeantibodies (Ab2) and anti anti-idiotype antibodies (Ab3). Ab2 arespecific for the target to which anti-FANCD2 antibodies of the inventionbind and are similar to FANCD2 antibodies in their binding specificitiesand biological activities [see, e.g., M. Wettendorff et al., “Modulationof anti-tumor immunity by anti-idiotypic antibodies.” In IdiotypicNetwork and Diseases, ed. by J. Cerny and J. Hiernaux, 1990 J 4177. Soc.Microbiol., Washington D.C.: pp. 203-229]. These anti-idiotype andanti-anti-idiotype antibodies are produced using techniques well knownto those of skill in the art. Such anti-idiotype antibodies (Ab2) canbear the internal image of FANCD2 and are thus useful for the samepurposes as FANCD2.

In general, polyclonal antisera, monoclonal antibodies and otherantibodies which bind to FANCD2 as the antigen (Ab1) are useful toidentify epitopes of FANCD2 to separate FANCD2 and its analogs fromcontaminants in living tissue (e.g., in chromatographic columns and thelike), and in general, as research tools and as starting materialessential for the development of other types of antibodies describedabove. Anti-idiotype antibodies (Ab2) are useful for binding the sametarget and thus are used in place of FANCD2 to induce useful ligands toFANCD2. The Ab3 antibodies are useful for the same reason the Ab 1 areuseful. Other uses, as research tools and as components for separationof FANCD2 from other contaminants, for example, are also contemplatedfor the above-described antibodies.

Antibodies that Bind Specifically to Monoubiquitinated FANCD2

The total cellular level of FANCD2 protein does not significantly changein response to DNA damage. Rather, DNA damage results inmonoubiquitination of FANCD2, as well as recruitment intoFANCD2-containing foci. It will be appreciated by one skilled in the artthat an alternative to measuring the presence of FANCD2-containing fociis to use a ligand which specifically binds the monoubiquitinated, butnot the unubiquitinated form of FANCD2. To detect the presence ofmonoubiquitinated FANCD2, the ligand is preferably associated with adetectable label as described above. The main advantage of using such aligand, as will be appreciated by one skilled in the art, is that, dueto the typically low basal level of monoubiquitinated FANCD2 in cellswith undamaged DNA, the level of FANCD2-containing foci can be measuredin a sample taken from a living subject using the level ofmonoubiquitinated FANCD2 as a surrogate marker by additional meansbesides immunofluorescence microscopy. An antibody which specificallyrecognizes the monoubiquitinated form of FANCD2 (FANCD2-L) hasconsiderable utility as a rapid diagnostic. For instance, this antibodycould be used for:

-   1) Immunohistochemistry (1H). This antibody could be used to examine    tissue sections prepared from solid tumors (e.g., breast, ovarian,    lung tumors). A positive signal by IH would predict that the tumor    will be resistant to cisplatin and related drugs.-   2) FACS analysis. Peripheral blood lymphocytes (PBLs) could be    screened with this antibody. A positive signal suggests the presence    of activated FANCD2, consistent with a recent exposure of an    individual to IR. or toxin. Thus, this antibody is a useful    extension of the radiation dosimeter assay described in this    application.-   3) A high-throughput assay to screen for inhibitors of the purified    FA complex (monoubiquitin ligase). These inhibitors will block the    ability of the FA complex to monoubiquitinate FANCD2 in vitro. The    new monoclonal antibody will be a useful reagent for end product    detection. Additional methods of measuring FANCD2-containing foci    using a ligand which specifically recognizes monoubiquitinated    FANCD2 include immunoblot analysis, or Enzyme linked immunosorbant    assays (ELISA) using extracts of samples collected from living    subjects, or FACS analysis (Harlow et al, 1999, Using Antibodies: A    Laboratory Manual, Cold Spring Harbor Laboratory Press, NY).

A sensitive measure of IR exposure is the increased monoubiquitinationof FANCD2. In undamaged cells, the (L/S) ratio of FANCD2-L(monoubiquitinated isoform) to FANCD2-S (unubiquitinated isoform) isapproximately 0.5-0.6. This ratio is readily calculated by comparing thedensity of the L band to the S band on a western blot. A sensitiveindicator of increased FANCD2 monoubiquitination and IR exposure is theconversion of the L/S ratio to 1.0 or greater.

Detectable Labels Useful for the Invention

The detectable label useful according to the invention is selected fromthe group consisting of radioactive, enzymatic, colorimetric,chemiluminescent, fluorescent, electrochemical labels and combinationsthereof. In a preferred embodiment of this aspect, the detectable labelis a fluorescent compound. In a particularly preferred embodiment, thedetectable label is selected from the group consisting of fluorescein,rhodamine, bodipy, cyanine, Alexa, Naphthofluorescein, Oregon Green,coumarin, dansyl, Texas Red, pyrene, Cascade Blue, and Alexa 350 andderivatives thereof. In certain embodiments, fluorescent proteins may beused. For example, green fluorescent proteins (GFPs) of cnidarians,which act as their energy-transfer acceptors in bioluminescence, can beused in the invention. A green fluorescent protein, as used herein, is aprotein that fluoresces green light, and a blue fluorescent protein is aprotein that fluoresces blue light. GFPs have been isolated from thePacific Northwest jellyfish, Aequorea victoria, from the sea pansy,Renilla renjformis, and from Phialidium gregarium (Ward et al., 1982,Photochem. Photobiol. 35:803-808; Levine et al., 1982, Comp. Biochem.Physiol. 72B:77-85).

A preferred fluorescent protein is green fluorescent protein (GFP) or amodified GFP. Wild-type GFP has long been used in the art. Starting fromgreen fluorescent protein, many modified versions have been derived withaltered or enhanced spectral properties as compared with wild-type GFP.See, e.g., U.S. Pat. No. 5,625,048; International Patent Publication WO97/28261; International Patent Publication WO 96/23810. Useful are themodified GFPs W1B and TOPAZ, available commercially from AuroraBiosciences Corp., San Diego, Calif. W1B contains the following changesfrom the wild-type GFP sequence: F64L, S65T, Y66W7 N1461, M153T, andV163A (see Table 1, page 519, of Tsien, 1998, Ann. Rev. Biochem.67:509-544). TOPAZ contains the following changes from the wild-type GFPsequence: S65G, V68L, S72A, and T203Y (see Table 1, page 519, of Tsien,1998, Ann. Rev. Biochem. 67:509-544). Wild-type nucleotide and aminoacid sequences of GFP are shown in FIG. 1 and SEQ ID NO: 1 ofInternational Patent Publication WO 97/28261; in FIG. 1 of Tsien, 1998,Ann. Rev. Biochem. 67:509544; and in Prasher et al., 1992, Gene 111:229.Of particular interest in using fluorescent proteins in FRET-basedscreening assays are variants of the A. Victoria GFP known as Cyan FP(CFP, Donor (D)) and Yellow FP (YFP, Acceptor (A)). As an example, theYFP variant can be made as a fusion protein with FANCD2 polypeptide.Vectors for the expression of GFP variants as fusions (Clontech) as wellas flurophore-labeled compounds (Molecular Probes) are known in the art.When expressing GFPs in mammalian cells, it may be advantageous toconstruct versions of the GFPs having altered codons that conform tothose—20 codons preferred by mammalian cells (Zolotukhin et al., J.Virol. 1996, 70:4646-46754; Yang et al., 1996, Nucl. Acids Res.24:4592-4593). Another way of improving GFP expression in mammaliancells is to provide an optimal ribosome binding site by the use of anadditional codon immediately after the starting methionine (Crarneli etal., 1996, Nature Biotechnology 14:315-319).

Diagnostic Methods According to the Invention

A diagnostic method of the invention comprises contacting a sample takenfrom a living subject, where the sample is preferably immobilized orfixed on a surface such as a microscope slide, with a ligand that bindsto human FANCD2. Such ligands are discussed in detail above, and arepreferably associated with a detectable label which provides a signal.The sample is then examined for the presence of signal concentrated innuclear foci of FANCD2 in the cells of the sample. The presence ofFANCD2-containing foci in the sample are then detected usingimmunofluorescence microscopy. In one embodiment, FANCD2 containing fociare detected by detecting a label on a primary antibody that binds toFANCD2. In another embodiment, FANCD2 containing foci are detected bydetecting the label of on a secondary antibody or reagent which binds toa primary antibody that binds to FANCD2. The presence ofFANCD2-containing foci in a sample is indicative of exposure of thesubject to a genotoxic agent, while the presence of diffuse signal isindicative of a lack of DNA damage in the sample. In addition, thenumber, size, and persistence of FANCD2-containing foci within a sample,as compared to a control sample, are directly proportional to the amountof DNA damage the cell has sustained. The best method to quantify toxinor radiation exposure is to measure the increase in the percentage ofcells with five or more bright foci. So, a population of peripheralblood lymphocytes from (unexposed) adult human controls has only 1-2%foci-positive cells (i.e., cells with greater than five FANCD2foci/cell). Following radiation exposure, there is an increase in thepercentage of foci-positive cells (to >10% of the cell population)depending on the dose and site of x-ray exposure.

The formation of foci is known to be time-dependent. It is preferablefor the sample to be collected within a relatively short time afterexposure or suspected exposure of the subject with a potential genotoxicagent. In a particular embodiment, samples are collected no longer than7 days after contact exposure. For example, samples are collected 1 hr,2 hrs, 4 hrs, 6 hrs, 12 hrs, 18 hrs, 24 hrs, 48 hrs, 72 hrs, 96 hrs, 120hrs, and 144 hrs and 168 hrs. In a particularly preferred embodiment,the samples are collected within 72 hours after exposure. In a mostpreferred embodiment, the samples are collected between 6 and 48 hoursafter exposure.

It will be appreciated by those skilled in the art that the presence ofa control sample will provide additional uses in quantitation ofexposure to genotoxic agents. Thus, in a preferred embodiment of thisaspect of the invention, the diagnostic method of this invention furthercomprises contacting the sample taken from a living subject, where thesample is preferably immobilized or fixed on a surface such as amicroscope slide, with a ligand that binds to human FANCD2. The sampleis then examined for the presence of signal concentrated in nuclear fociof FANCD2 in the cells of the sample. The examining step is any suitableassay step, including, without limitation, fluorescentimmunofluorescence microscopy or immunohistochemical analysis. Thepresence of FANCD2-containing foci in a test sample as compared to acontrol sample is indicative of exposure of the subject to a genotoxicagent, while the presence of diffuse signal is indicative of a lack ofDNA damage in the sample. In addition, a difference in the number, size,and persistence of FANCD2-containing foci within a sample relative tocontrol sample is indicative of a difference in DNA damage in responseto exposure to a genotoxic agent, and the degree of foci formationrelative to a reference sample is indicative of a different extent ofDNA damage in response to said exposure.

Pre- and Post-Therapy Diagnostic Methods

The invention provides for further methods for determining thesensitivity of a patient to radiotherapy or chemotherapy. In oneembodiment, the diagnostic method comprises exposing a subject orextracted lymphocytes from a subject, prior to chemotherapy orradiotherapy, to a low dose of a genotoxic agent, contacting the sampletaken from the living subject with a ligand that binds to human FANCD2,where the sample is preferably immobilized or fixed on a surface such asa microscope slide. Such ligands are discussed in detail above, and arepreferably associated with a detectable label which provides a signal,also as discussed above. The sample is then examined for the presence ofsignal concentrated in nuclear foci of FANCD2 in the cells of thesample. The examining step is any suitable assay step, including,without limitation, immunofluorescence microscopy or immunohistochemicalanalysis. Reduced formation of FANCD2-containing foci compared to acontrol indicates radio- or chemoresistance. In contrast, enhanced fociformation may indicate relative radio- or chemosensitivity.Alternatively, samples can be contacted with a ligand which bindsspecifically to monoubiquitinated FANCD2. Thus, in another embodiment ofthis aspect of the invention, the diagnostic method of this inventioncomprises exposing a patient to a low dose of genotoxic agent anddetecting the presence of FANCD2-containing foci relative to a controlsample, wherein the presence of foci formation relative to a controlsample is indicative of a difference in sensitivity of said patient togenotoxic agent.

Additionally, this method can be employed to rapidly and readily assessDNA damage in patients treated with gamma irradiation or otherchemotherapeutic agents, particularly those known to cause DNA damage.Therefore, a further embodiment of this invention is a diagnostic methodof determining the extent of DNA damage caused by exposure of a subjectto therapeutic agent, comprising collecting a sample from said patientfollowing exposure and detecting the presence of FANCD2-containing focirelative to a control sample. A difference in foci formation relative tosaid control sample is indicative of a difference in DNA damage inresponse to said exposure, and wherein the degree of foci formationrelative to a control sample is indicative of a different extent of DNAdamage in response to said exposure.

Diagnostic Methods Using Samples Collected from Subjects

Prior to administering radiation therapy or chemotherapy to a subjectwho has cancer, it would be advantageous to be able to pre-test a tumorsample to ascertain its sensitivity towards a therapeutic agent such asionizing radiation or cisplatin. Such a pre-test will ensure that thebest mode of therapy is used for the particular cancer. Thus, in oneaspect of the invention, a method of determining the sensitivity of asample towards genotoxic agents is provided, comprising the followingsteps: collecting a sample from the patient, exposing the sample ortissue to a genotoxic agent, and detecting the presence ofFANCD2-containing foci relative to a control sample. The sample cancomprise a tumor biopsy taken from a living subject. A difference infoci formation relative to the control sample is indicative of adifference in DNA damage in response to the exposure, and the degree offoci formation relative to a control sample is indicative of a differentsensitivity of the tissue towards the agent. Any therapeutic agent whichis known to cause genotoxic stress can be tested in this method.Examples of such genotoxic agents include ionizing radiation orcisplatin.

In certain situations, it may be important to monitor the progress of atumor sample in the subject over a period of time. For example, while asubject is receiving chemotherapy for a tumor, it would be important todetermine whether the tumor has developed resistance towards thetherapeutic agent. Therefore, in another embodiment, the method furthercomprises collecting multiple samples from a subject at different timepoints. These collected samples are exposed to the same genotoxic agent,and are preferably exposed to the same level of the genotoxic agent.FANCD2-containing foci are then detected in these samples using any ofthe methods previously described. By comparing the FANCD2-containingfoci in these samples to control samples, and by further monitoring thechange in FANCD2-containing foci in these samples over time, it can beascertained whether the tissue's sensitivity towards a particulargenotoxic agent has changed. For example, a decrease inFANCD2-containing foci over time indicates a loss in sensitivity of thesample towards that particular genotoxic agent.

Drug Screening Methods of the Invention

High throughput screening has been a frequent first step used inindustry and academia for the identification of compounds that targetspecific molecules or cellular processes. As described below, themonitoring of FANCD2 foci in samples, in vitro and in vivo, provides auseful screening tool for new drug discovery. The invention provides formethods for screening for a radioprotective agent.

-   1) A radioprotective agent may, itself, cause an increase in FANCD2    foci in peripheral blood lymphocytes. By enhancing the baseline    level of FANCD2 foci, the radioprotective agent may activate low    (baseline) levels of DNA repair. If an individual is treated with a    radioprotective agent and is subsequently exposed to a genotoxic    agent, an individual may have an increased (primed) DNA repair    response, thus leading to enhanced protection from the radiation    damage.-   2) In another embodiment, a putative radioprotective agent may    enhance DNA repair, without increasing FANCD2 foci.    FANCD2-containing foci are monitored following exposure to low doses    of a genotoxic agent may allow an investigator to assess the    relative protective effect of putative agent. For instance, in this    case, radioprotection by a novel agent will result in decreased    FANCD2 following challenge with a genotoxic agent.

Methods of screening test compounds are described which are useful inidentifying compositions that are active in FANCD2-containing fociformation. Such a composition may be useful as a protective agentagainst a genotoxic agent or as a chemosensitizer. These methodscomprise contacting a biological sample with test compound and detectingthe presence of FANCD2 foci formation relative to a control sample. Adifference in the degree of foci formation relative to a control sampleis indicative of an agent active in foci formation.

One embodiment of this method further comprises employing a FANCD2ligand associated with a detectable label to detect FANCD2-containingfoci. In this embodiment, such a screening method is employed toidentify agents which inhibit the formation of FANCD2-containing foci.According to this method, a selected biological sample is contacted witha test compound (i.e., the “test sample”) as well as an identical samplewithout test compound (i.e., the “control sample”) under conditionswhich normally result in formation of FANCD2-containing foci, forexample, by contact with a genotoxic agent. In this aspect, the level ofgenotoxic agent is selected such that a relatively high number of fociare reproducibly formed in a control sample. The test sample and controlsample are then contacted with a FANCD2 ligand which is associated witha detectable label. Alternatively, samples may be contacted with aligand which specifically recognizes monoubiquitinated FANCD2. The testsample and control sample are subsequently examined and compared for thepresence, number and size of FANCD2-containing foci using methods suchas immunofluorescence microscopy. A reduction in, or absence of,FANCD2-containing foci in the test sample relative to the control sampleindicates that the test compound is capable of inhibiting the formationof FANCD2-containing foci in this assay. The presence and/or number offoci are indicated by the level or intensity of the signal generated bythe label on the ligand. The signal (or its level of expression orintensity) indicates the presence and number of FANCD2-containing foci.When the signals generated by the label in the test sample are comparedwith the signals (if any) generated by the labels in the control sample,a lesser detectable signal in the test cell indicates that said testcompound has inhibited the formation of FANCD2-containing foci in thecell. For example, test samples exhibiting at least 10% less, forexample 10%, 20%, 30%, 50%, 75%, or up to 100% less, FANCD2-containingfoci when compared to a control sample is indicative of an agent activein inhibiting the formation of FANCD2-containing foci in the cell.

In another aspect of this embodiment, such a screening method isemployed to identify agents which enhance the formation ofFANCD2-containing foci. Such a method involves contacting a selectedbiological sample with a test compound (i.e., the “test sample”) as wellas an identical sample without test compound (i.e., the “controlsample”). The test sample and control sample may optionally be exposedto a low level of genotoxic agent. The test sample and control sampleare subsequently examined and compared for the presence and number ofFANCD2-containing foci using methods described above. An increase of atleast 10%, for example 10%, 20%, 30%, 50%, 75%, 100%, 200%, or 500%, inFANCD2-containing foci in the test sample relative to the control sampleindicates that the test compound is capable of inducing the formation ofFANCD2-containing foci in this assay.

Mutagenicity Testing for FANCD2-Foci Inducing Compounds

Upon identification of such compounds active in formation ofFANCD2-containing foci, it will be necessary to determine whether thecompounds posses genotoxic activity, or merely induce FANCD2-containingfoci without possessing genotoxicity. It will be appreciated by oneskilled in the art that genotoxicity can be measured using one of manywell-established assays. A comprehensive list of such methods can befound in Toxicological Principles for the Safety Assessment of DirectFood Additives and Color Additives Used in Food. Draft Redbook II.Washington, D.C.: CFSAN, Food and Drug Administration, which is alsoavailable in electronic form (on the world wide web atwww.cfsan.fda.gov/˜redbook/red-toca.html). Common methods include theAmes test for mutagenicity (Ames, B. N., McCann, J. & Yamasaki, E.(1975) Methods for detecting carcinogens and mutagens withSalmonella/mammalian-microsome mutagenicity test. Mutation Res. 31,347-364.), chromosome aberration test (Galloway, S. M., Aardema, M. J.,Ishidate Jr., M., Ivett, J. L., Kirkland, D. J., Morita, T., Mosesso,P., and Sofuni, T. (1994). Report from working group on in vitro testsfor chromosomal aberrations. Mutation Research 312, 241-261), which areincorporated in its entirety by reference.

Briefly, the Ames test is performed using the following procedure. Theobjective of this assay is to evaluate the mutagenic potential of testchemicals by studying their effect on one or more histidine requiringstrains of Salmonella typhimurium in the absence and presence of a livermetabolizing system. When the cultures are exposed to a mutagen some ofthe bacteria undergo genetic changes due to chemical interactionsresulting in reversion of the bacteria to a non-histidine-requiringstate. The reverted bacteria will then grow in the absence of exogenoushistidine thus providing an indication of the potential of the chemicalto cause mutation. Multiple tester strains are necessary becausedifferent strains are mutated by a different class (or differentclasses) of compound. Other types of bacteria can also be used, e.g.tryptophan requiring strains of Escherichia coli. The basis of the testis very similar, the only difference being that the bacteria have arequirement for a different amino acid. Nutrient broth is inoculatedwith the appropriate Salmonella strain (TA98, TA100, TA1535, TA1537,TA102) and incubated overnight. A dose rangefinder for the test chemicalis carried out using strain TA100 only over a wide dose range. Bacterialculture, test chemical and S9 mix (or co-factor solution) are mixed withsoft agar and then added to minimal agar plates. The plates areincubated, and inverted in the dark, for 48-72 hours. After this timethe number of revertant colonies are counted. An additional two mutationexperiments are carried out, with doses chosen on the basis of therangefinder. The number of colonies are counted from both experimentsand the mean is calculated for the individual plate counts for each dosewithin an experiment. Statistical analysis of the counts are carriedout, and the results for mutagenicity are assessed. Compounds testedshould ideally form less than 10-fold higher, for example 10-fold,8-fold, 5-fold, 2-fold, 1-fold higher, revertants at 1 μM of compoundwhen compared to control samples that were not exposed to mutagen.

GFP-FANCD2-Based Screening Methods

In yet another embodiment, a screening method comprises detectingFANCD2-containing foci in cells expressing genetic constructs expressingFANCD2 fused to a reporter fluorescent protein, and using fluorescencemicroscopy. Recombinant nucleic acid constructs of particular use in theinvention include those which comprise in-frame fusions of sequencesencoding the human FANCD2 or fragments thereof and a fluorescentprotein. Such a nucleic acid molecule encodes a polypeptide comprisingFANCD2, fused to a fluorescent protein label and operatively linked togene regulatory sequences.

This embodiment of the screening method further comprises cellstransfected with a recombinant nucleic acid construct encoding the humanFANCD2 protein fused to a reporter protein. This embodiment of thescreening method differs from the previous embodiment in the method ofdetection. In this embodiment, FANCD2-containing foci is detectedwithout the need for a FANCD2-binding ligand. Instead FANCD2-containingfoci are detected directly by detecting the reporter protein which isfused to FANCD2.

As taught in Examples 4 and 5, a preferred genetic construct accordingto this invention employs the enhanced green fluorescent protein (eGFP)fused to the N-terminal methionine of full-length human FANCD2. PD20cells expressing this genetic construct express the fusion protein whichis monoubiquitinated upon exposure of cells to genotoxic agents.Furthermore, the GFP-FANCD2 construct corrects the hypersensitivity ofthese cells towards genotoxic agents such as Mitomycin C (MMC). Exposureof these cells with genotoxic agents results in formation ofFANCD2-containing foci, as visualized by detecting the eGFPfluorescence, obviating the need for ligands which bind FANCD2 ormonoubiquitinated FANCD2.

FRET-Based Screening Methods

In a still further embodiment, FANCD2-containing foci are detected incells expressing two genetic constructs, wherein at least one constructexpresses FANCD2 fused to a first reporter fluorescent protein, and asecond construct expresses a second protein associated withFANCD2-containing foci fused to a second reporter fluorescent protein,and wherein FANCD2-containing foci are detected by fluorescenceresonance energy transfer (FRET). Preferred examples of proteinsassociated with FANCD2-containing foci include NBS1 (Nakanishi et al.,2002. Nat Cell Biol. 4:913-20) and BRCA1 (Garcia-Higuera et al., 2001.Mol Cell. 7:249-62). In this embodiment, two fluorescent reporterproteins are chosen which serve as acceptor and donor fluorophores. Uponexposure of cells to genotoxic agents, the FANCD2-fluorescent reporterfusion protein and either the NBS1-fluorescent reporter fusion proteinor BRCA1-fluorescent reporter fusion protein associate to formFANCD2-containing foci. In one embodiment, constructs are generated, oneof which encodes FANCD2 fused with a donor fluorescent protein such asthe cyan variant of GFP (CFP). Another construct is generated whichencodes the acceptor fluorescent protein, preferably the yellow variantof GFP (YFP), fused with a foci-associated protein selected from thegroup consisting of NBS1, Histone 2AX, and BRCA1. These constructs areoperatively linked with promoter and terminator sequences in a vector asdescribed previously, or by employing methods well known in the art. Thetwo fluorescent protein fusion constructs can be placed on separatevectors or on the same vector. A host cell is then transformed such thatboth constructs are expressed. Expression of both constructs can betested by detecting fluorescence of CFP and YFP in the presence orabsence of exposure to genotoxic agents using methods known in the art,for example by fluorescence microscopy at the appropriate wavelengths.

The physical proximity of the two fluorescent proteins results inincreased fluorescence resonance energy transfer, which can be detectedusing fluorescent methods, including FRET microscopy, ratio imaging, orratiometric fluorimetry. An instrument such as FLIPR™ can be set toalternate between reading signals at two different wavelengths with acycling time of about one second, and is therefore extremely useful inmeasuring samples in high-throughput.

Fluorescence resonance energy transfer (FRET) is a non-radiative processwhereby energy from a fluorescent donor molecule is transferred to anacceptor molecule without the involvement of a photon. Excitation of thedonor molecule enhances the fluorescence emission of thelonger-wavelength acceptor molecule (i.e., sensitized acceptoremission). The quantum yield of the donor fluorescence emission isconcomitantly diminished. FRET has become a valuable tool formicroscopy, because the efficiency of energy transfer has a stronginverse dependence on the distance between the donor and acceptormolecules. Thus, the appearance of FRET is a highly specific indicatorof the proximity of the two molecules. This has led to the use of FRETefficiency as a “spectroscopic ruler” to measure molecular distances.

The recent availability of green fluorescent protein (GFP) mutants withshifted excitation and emission spectra has made it feasible to measureprotein-protein interactions by using GFP tags as intracellular markers.GFP-tagged protein chimeras are expressed intracellularly and do notrequire any chemical treatment to become fluorescent. FRET can alsooccur between fusions of blue-emitting and green-emitting GFP variants.

As described above, a donor fluorescent protein label is capable ofabsorbing a photon and transferring energy to another fluorescent label.The acceptor fluorescent protein label is capable of absorbing energyand emitting a photon. If needed, the linker connects the bindingdomain, sequence or polypeptide either directly, or indirectly throughan intermediary linkage, with one or both of the donor and acceptorfluorescent protein labels or the fluorescent label and, optionally, thequencher if a non-FRET assay is being performed. Regardless of therelative order of the binding domain, sequence or polypeptide or itsbinding partner and the donor and acceptor fluorescent protein labels ona polypeptide molecule, it is essential that sufficient distance beplaced between the donor and acceptor or the fluorescent label andcorresponding quencher by the linker and/or the binding domain,sequence, nucleic acid or polypeptide and corresponding binding partnerto ensure that FRET does not occur unless the binding domain, sequenceor polypeptide and its binding partner bind. It is desirable, asdescribed in greater detail in WO97/28261, to select a donor fluorescentprotein label with an emission spectrum that overlaps with theexcitation spectrum of an acceptor fluorescent protein label. In someembodiments of the invention the overlap in emission and excitationspectra will facilitate FRET. A fluorescent protein of use in theinvention includes, in addition to those with intrinsic fluorescentproperties, proteins that fluoresce due to intramolecular rearrangementsor the addition of cofactors that promote fluorescence.

For example, green fluorescent proteins (GFPs) of cnidarians, which actas their energy-transfer acceptors in bioluminescence, can be used inthe invention. A green fluorescent protein, as used herein, is a proteinthat fluoresces green light, and a blue fluorescent protein is a proteinthat fluoresces blue light. GFPs have been isolated from the PacificNorthwest jellyfish, Aequorea victoria, from the sea pansy, Renillarenjformis, and from Phialidium gregarium (Ward et al., 1982, Photochem.Photobiol. 35:803-808; Levine et al., 1982, Comp. Biochem. Physiol.72B:77-85). A variety of Aequorea-related GFPs having useful excitationand emission spectra have been engineered by modifying the amino acidsequence of a naturally-occurring GFP from Aequorea Victoria (Prasher etal., 1992, Gene 111:229-233; Heim et al., 1994, Proc. Natl. Acad. Sci.U.S.A. 91:12501-12504; PCTUS95/14692). As used herein, a fluorescentprotein is an Aequorea-related fluorescent protein if any contiguoussequence of 150 amino acids of the fluorescent protein has at least 85%sequence identity with an amino acid sequence, either contiguous ornon-contiguous, from the wild-type Aequorea green fluorescent protein(SwissProt Accession No. P42212). Similarly, the fluorescent protein maybe related to Renilla or Phialidium wild-type fluorescent proteins usingthe same standards. Aequorea-related fluorescent proteins include, forexample, wild-type (native) Aequorea victoria GFP, whose nucleotide anddeduced amino acid sequences are presented in GenBank Accession Nos.L29345, M62654, M62653 and other Aequorea-related engineered versions ofGreen Fluorescent Protein, of which some are listed above. Several ofthese, i.e., P4, P4-3, W7 and W2 fluoresce at a distinctly shorterwavelength than wild type.

Recombinant nucleic acid molecules encoding single- or tandemfluorescent protein/polypeptide comprising engineered binding domain,sequences or polypeptides or their binding partners useful in theinvention may be expressed for in vivo assays of the activity of amodifying enzyme on the encoded products.

Similar assays using different ligands, different detection techniques,etc. are readily designed by one of skill in the art using theinformation provided in the art generally.

Vectors Useful for the Invention

There is a wide array of vectors known and available in the art that areuseful for the expression of differentially expressed nucleic acidmolecules according to the invention. The selection of a particularvector clearly depends upon the intended use the polypeptide encoded bythe differentially expressed nucleic acid. For example, the selectedvector must be capable of driving expression of the polypeptide in thedesired cell type, whether that cell type be prokaryotic or eukaryotic.Many vectors comprise sequences allowing both prokaryotic vectorreplication and eukaryotic expression of operably linked gene sequences.

Vectors useful according to the invention may be autonomouslyreplicating, that is, the vector, for example, a plasmid, existsextrachromosomally and its replication is not necessarily directlylinked to the replication of the host cell's genome. Alternatively, thereplication of the vector may be linked to the replication of the host'schromosomal DNA, for example, the vector may be integrated into thechromosome of the host cell as achieved by retroviral vectors.

Vectors useful according to the invention preferably comprise sequencesoperably linked to the differentially expressed sequences that permitthe transcription and translation of the sequence. Sequences that permitthe transcription of the linked differentially expressed sequenceinclude a promoter and optionally also include an enhancer element orelements permitting the strong expression of the linked sequences. Theterm “transcriptional regulatory sequences” refers to the combination ofa promoter and any additional sequences conferring desired expressioncharacteristics (e.g., high level expression, inducible expression,tissue- or cell-type-specific expression) on an operably linked nucleicacid sequence.

The selected promoter may be any DNA sequence that exhibitstranscriptional activity in the selected host cell, and may be derivedfrom a gene normally expressed in the host cell or from a gene normallyexpressed in other cells or organisms. Examples of promoters include,but are not limited to the following: A) prokaryotic promoters—E. colilac, tac, or trp promoters, lambda phage PR or PL promoters,bacteriophage T7, T3, Sp6 promoters, B. subtilis alkaline proteasepromoter, and the B. stearothermophilus maltogenic amylase promoter,etc.; B) eukaryotic promoters—yeast promoters, such as GAL1, GAL4 andother glycolytic gene promoters (see for example, Hitzeman et al., 1980,J. Biol. Chem. 255: 12073-12080; Alber & Kawasaki, 1982, J. Mol. Appl.Gen. 1: 419-434), LEU2 promoter (Martinez-Garcia et al., 1989, Mol GenGenet. 217: 464-470), alcohol dehydrogenase gene promoters (Young etal., 1982, in Genetic Engineering of Microorganisms for Chemicals,Hollaender et al., eds., Plenum Press, NY), or the TPI1 promoter (U.S.Pat. No. 4,599,311); insect promoters, such as the polyhedrin promoter(U.S. Pat. No. 4,745,051; Vasuvedan et al., 1992, FEBS Lett. 311: 7-11),the P10 promoter (Vlak et al., 1988, J. Gen. Virol. 69: 765-776), theAutographa californica polyhedrosis virus basic protein promoter (EP397485), the baculovirus immediate-early gene promoter gene 1 promoter(U.S. Pat. Nos. 5,155,037 and 5,162,222), the baculovirus 39Kdelayed-early gene promoter (also U.S. Pat. Nos. 5,155,037 and5,162,222) and the OpMNPV immediate early promoter 2; mammalianpromoters—the SV40 promoter (Subramani et al., 1981, Mol. Cell. Biol. 1:854-864), metallothionein promoter (MT-1; Palmiter et al., 1983, Science222: 809-814), adenovirus 2 major late promoter (Yu et al., 1984, Nucl.Acids Res. 12: 9309-21), cytomegalovirus (CMV) or other viral promoter(Tong et al., 1998, Anticancer Res. 18: 719-725), or even the endogenouspromoter of a gene of interest in a particular cell type.

A selected promoter may also be linked to sequences rendering itinducible or tissue-specific. For example, the addition of atissue-specific enhancer element upstream of a selected promoter mayrender the promoter more active in a given tissue or cell type.Alternatively, or in addition, inducible expression may be achieved bylinking the promoter to any of a number of sequence elements permittinginduction by, for example, thermal changes (temperature sensitive),chemical treatment (for example, metal ion- or IPTG-inducible), or theaddition of an antibiotic inducing agent (for example, tetracycline).

Regulatable expression is achieved using, for example, expressionsystems that are drug inducible (e.g., tetracycline, rapamycin orhormone-inducible). Drug-regulatable promoters that are particularlywell suited for use in mammalian cells include the tetracyclineregulatable promoters, and glucocorticoid steroid-, sex hormonesteroid-, ecdysone-, lipopolysaccharide (LPS)- andisopropylthiogalactoside (IPTG)-regulatable promoters. A regulatableexpression system for use in mammalian cells should ideally, but notnecessarily, involve a transcriptional regulator that binds (or fails tobind) non-mammalian DNA motifs in response to a regulatory agent, and aregulatory sequence that is responsive only to this transcriptionalregulator.

Tissue-specific promoters may also be used to advantage indifferentially expressed sequence-encoding constructs of the invention.A wide variety of tissue-specific promoters is known. As used herein,the term “tissue-specific” means that a given promoter istranscriptionally active (i.e., directs the expression of linkedsequences sufficient to permit detection of the polypeptide product ofthe promoter) in less than all cells or tissues of an organism. A tissuespecific promoter is preferably active in only one cell type, but may,for example, be active in a particular class or lineage of cell types(e.g., hematopoietic cells). A tissue specific promoter useful accordingto the invention comprises those sequences necessary and sufficient forthe expression of an operably linked nucleic acid sequence in a manneror pattern that is essentially the same as the manner or pattern ofexpression of the gene linked to that promoter in nature. The followingis a non-exclusive list of tissue specific promoters and literaturereferences containing the necessary sequences to achieve expressioncharacteristic of those promoters in their respective tissues; theentire content of each of these literature references is incorporatedherein by reference. Examples of tissue specific promoters useful in thepresent invention are as follows:

Bowman et al., 1995 Proc. Natl. Acad. Sci. USA 92, 12115-12119 describea brain-specific transferrin promoter; the synapsin I promoter is neuronspecific (Schoch et al., 1996 J. Biol. Chem. 271, 3317-3323); the nestinpromoter is post-mitotic neuron specific (Uetsuki et al., 1996 J. Biol.Chem. 271, 918-924); the neurofilament light promoter is neuron specific(Charron et al., 1995 J. Biol. Chem. 270, 30604-30610); theacetylcholine receptor promoter is neuron specific (Wood et al., 1995 J.Biol. Chem. 270, 30933-30940); and the potassium channel promoter ishigh-frequency firing neuron specific (Gan et al., 1996 J. Biol. Chem271, 5859-5865). Any tissue specific transcriptional regulatory sequenceknown in the art may be used to advantage with a vector encoding adifferentially expressed nucleic acid sequence obtained from an animalsubjected to pain.

In addition to promoter/enhancer elements, vectors useful according tothe invention may further comprise a suitable terminator. Suchterminators include, for example, the human growth hormone terminator(Palmiter et al., 1983, supra), or, for yeast or fungal hosts, the TPI1(Alber & Kawasaki, 1982, supra) or ADH3 terminator (McKnight et al.,1985, EMBO J. 4: 2093-2099).

Vectors useful according to the invention may also comprisepolyadenylation sequences (e.g., the SV40 or Ad5E1b poly(A) sequence),and translational enhancer sequences (e.g., those from Adenovirus VARNAs). Further, a vector useful according to the invention may encode asignal sequence directing the recombinant polypeptide to a particularcellular compartment or, alternatively, may encode a signal directingsecretion of the recombinant polypeptide.

a. Plasmid Vectors.

Any plasmid vector that allows expression of a differentially expressedcoding sequence of the invention in a selected host cell type isacceptable for use according to the invention. A plasmid vector usefulin the invention may have any or all of the above-noted characteristicsof vectors useful according to the invention. Plasmid vectors usefulaccording to the invention include, but are not limited to the followingexamples: Bacterial—pQE70, pQE60, pQE-9 (Qiagen) pBs, phagescript,psiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a(Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5(Pharmacia); Eukaryotic—pWLneo, pSV2cat, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, and pSVL (Pharmacia). However, any other plasmid orvector may be used as long as it is replicable and viable in the host.

b. Bacteriophage Vectors.

There are a number of well known bacteriophage-derived vectors usefulaccording to the invention. Foremost among these are the lambda-basedvectors, such as Lambda Zap II or Lambda-Zap Express vectors(Stratagene) that allow inducible expression of the polypeptide encodedby the insert. Others include filamentous bacteriophage such as theM13-based family of vectors.

c. Viral Vectors.

A number of different viral vectors are useful according to theinvention, and any viral vector that permits the introduction andexpression of one or more of the differentially expressedpolynucleotides of the invention in cells is acceptable for use in themethods of the invention. Viral vectors that can be used to deliverforeign nucleic acid into cells include but are not limited toretroviral vectors, adenoviral vectors, adeno-associated viral vectors,herpesviral vectors, and Semliki forest viral (alphaviral) vectors.Defective retroviruses are well characterized for use in gene transfer(for a review see Miller, A. D. (1990) Blood 76:271). Protocols forproducing recombinant retroviruses and for infecting cells in vitro orin vivo with such viruses can be found in Current Protocols in MolecularBiology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates,(1989), Sections 9.10-9.14, and other standard laboratory manuals.

In addition to retroviral vectors, Adenovirus can be manipulated suchthat it encodes and expresses a gene product of interest but isinactivated in terms of its ability to replicate in a normal lytic virallife cycle (see for example Berkner et al., 1988, BioTechniques 6:616;Rosenfeld et al., 1991, Science 252:431-434; and Rosenfeld et al., 1992,Cell 68:143-155). Suitable adenoviral vectors derived from theadenovirus strain Ad type 5 d1324 or other strains of adenovirus (e.g.,Ad2, Ad3, Ad7 etc.) are well known to those skilled in the art.Adeno-associated virus (AAV) is a naturally occurring defective virusthat requires another virus, such as an adenovirus or a herpes virus, asa helper virus for efficient replication and a productive life cycle.(For a review see Muzyczka et al., 1992, Curr. Topics in Micro. andImmunol. 158:97-129). An AAV vector such as that described in Traschinet al. (1985, Mol. Cell. Biol. 5:3251-3260) can be used to introducenucleic acid into cells. A variety of nucleic acids have been introducedinto different cell types using AAV vectors (see, for example, Hermonatet al., 1984, Proc. Natl. Acad. Sci. USA 81: 6466-6470; and Traschin etal., 1985, Mol. Cell. Biol. 4: 2072-2081).

Host Cells

Any cell into which a recombinant vector carrying a gene encoding anucleic acid sequence differentially expressed in an animal subjected topain may be introduced and wherein the vector is permitted to drive theexpression of the peptide encoded by the differentially expressedsequence is useful according to the invention. Any cell in which adifferentially expressed molecule of the invention may be expressed andpreferably detected is a suitable host, wherein the host cell ispreferably a mammalian cell and more preferably a human cell. Vectorssuitable for the introduction of differentially expressed nucleic acidsequences to host cells from a variety of different organisms, bothprokaryotic and eukaryotic, are described herein above or known to thoseskilled in the art.

Host cells may be prokaryotic, such as any of a number of bacterialstrains, or may be eukaryotic, such as yeast or other fungal cells,insect or amphibian cells, or mammalian cells including, for example,rodent, simian or human cells. Cells may be primary cultured cells, forexample, primary human fibroblasts or keratinocytes, or may be anestablished cell line, such as NIH3T3, 293T or CHO cells. Further,mammalian cells useful in the present invention may be phenotypicallynormal or oncogenically transformed. It is assumed that one skilled inthe art can readily establish and maintain a chosen host cell type inculture.

Introduction of Vectors to Host Cells.

Vectors useful in the present invention may be introduced to selectedhost cells by any of a number of suitable methods known to those skilledin the art. For example, vector constructs may be introduced toappropriate bacterial cells by infection, in the case of E. colibacteriophage vector particles such as lambda or M13, or by any of anumber of transformation methods for plasmid vectors or forbacteriophage DNA. For example, standard calcium-chloride-mediatedbacterial transformation is still commonly used to introduce naked DNAto bacteria (Sambrook et al., 1989, Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.),but electroporation may also be used (Ausubel et al., 1988, CurrentProtocols in Molecular Biology, (John Wiley & Sons, Inc., NY, N.Y.)).

For the introduction of vector constructs to yeast or other fungalcells, chemical transformation methods are generally used (e.g. asdescribed by Rose et al., 1990, Methods in Yeast Genetics, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.). For transformationof S. cerevisiae, for example, the cells are treated with lithiumacetate to achieve transformation efficiencies of approximately 104colony-forming units (transformed cells)/μg of DNA. Transformed cellsare then isolated on selective media appropriate to the selectablemarker used. Alternatively, or in addition, plates or filters liftedfrom plates may be scanned for GFP fluorescence to identify transformedclones.

For the introduction of vectors comprising differentially expressedsequences to mammalian cells, the method used will depend upon the formof the vector. Plasmid vectors may be introduced by any of a number oftransfection methods, including, for example, lipid-mediatedtransfection (“lipofection”), DEAE-dextran-mediated transfection,electroporation or calcium phosphate precipitation. These methods aredetailed, for example, in Current Protocols in Molecular Biology(Ausubel et al., 1988, John Wiley & Sons, Inc., NY, N.Y.).

Lipofection reagents and methods suitable for transient transfection ofa wide variety of transformed and non-transformed or primary cells arewidely available, making lipofection an attractive method of introducingconstructs to eukaryotic, and particularly mammalian cells in culture.For example, Lipofectamine™ (Life Technologies) or LipoTaxi™(Stratagene) kits are available. Other companies offering reagents andmethods for lipofection include BioRad Laboratories, CLONTECH, GlenResearch, InVitrogen, JBL Scientific, MBI Fermentas, PanVera, Promega,Quantum Biotechnologies, Sigma-Aldrich, and Wako Chemicals USA.

Following transfection with a vector of the invention, eukaryotic (e.g.,human) cells successfully incorporating the construct (intra- orextrachromosomally) may be selected, as noted above, by either treatmentof the transfected population with a selection agent, such as anantibiotic whose resistance gene is encoded by the vector, or by directscreening using, for example, FACS of the cell population orfluorescence scanning of adherent cultures. Frequently, both types ofscreening may be used, wherein a negative selection is used to enrichfor cells taking up the construct and FACS or fluorescence scanning isused to further enrich for cells expressing differentially expressedpolynucleotides or to identify specific clones of cells, respectively.For example, a negative selection with the neomycin analog G418 (LifeTechnologies, Inc.) may be used to identify cells that have received thevector, and fluorescence scanning may be used to identify those cells orclones of cells that express the vector construct to the greatestextent.

Test Compounds According to the Invention

Whether in vitro or in an in vivo system, the invention encompassesmethods by which to screen compositions which may enhance or inhibit theformation of FANCD2-containing foci. Candidate modulator compounds fromlarge libraries of synthetic or natural compounds can be screened.Numerous means are currently used for random and directed synthesis ofsaccharide, peptide, and nucleic acid based compounds. Syntheticcompound libraries are commercially available from a number of companiesincluding Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex(Princeton, N.J.), Brandon Associates (Merrimack, N.H.), and Microsource(New Milford, Conn.). A rare chemical library is available from Aldrich(Milwaukee, Wis.). Combinatorial libraries are available and can beprepared. Alternatively, libraries of natural compounds in the form ofbacterial, fungal, plant and animal extracts are available from e.g.,Pan Laboratories (Bothell, Wash.) or MycoSearch (NC), or are readilyproducible by methods well known in the art. Additionally, natural andsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical, and biochemical means.

Useful compounds may be found within numerous chemical classes, thoughtypically they are organic compounds, including small organic compounds.Small organic compounds have a molecular weight of more than 50 yet lessthan about 2,500 Daltons, preferably less than about 750, morepreferably less than about 350 Daltons. Exemplary classes includeheterocycles, peptides, saccharides, steroids, and the like. Thecompounds may be modified to enhance efficacy, stability, pharmaceuticalcompatibility, and the like. Structural identification of an agent maybe used to identify, generate, or screen additional agents. For example,where peptide agents are identified, they may be modified in a varietyof ways to enhance their stability, such as using an unnatural aminoacid, such as a D-amino acid, particularly D-alanine, by functionalizingthe amino or carboxylic terminus, e.g., for the amino group, acylationor alkylation, and for the carboxyl group, esterification oramidification, or the like.

Candidate modulators which may be screened according to the methods ofthe invention include receptors, enzymes, ligands, regulatory factors,and structural proteins. Candidate modulators also include nuclearproteins, cytoplasmic proteins, mitochondrial proteins, secretedproteins, plasmalemma-associated proteins, serum proteins, viralantigens, bacterial antigens, protozoan antigens and parasitic antigens.Candidate modulators additionally comprise proteins, lipoproteins,glycoproteins, phosphoproteins and nucleic acids (e.g., RNAs such asribozymes or antisense nucleic acids). Proteins or polypeptides whichcan be screened using the methods of the present invention includehormones, growth factors, neurotransmitters, enzymes, clotting factors,apolipoproteins, receptors, drugs, oncogenes, tumor antigens, tumorsuppressors, structural proteins, viral antigens, parasitic antigens,bacterial antigens and antibodies (see below).

Candidate modulators which may be screened according to the inventionalso include substances for which a test cell or organism might bedeficient or that might be clinically effective in higher-than-normalconcentration as well as those that are designed to eliminate thetranslation of unwanted proteins. Nucleic acids of use according to theinvention not only may encode the candidate modulators described above,but may eliminate or encode products which eliminate deleteriousproteins. Such nucleic acid sequences are antisense RNA and ribozymes,as well as DNA expression constructs that encode them. Note thatantisense RNA molecules, ribozymes or genes encoding them may beadministered to a test cell or organism by a method of nucleic aciddelivery that is known in the art, as described below. Inactivatingnucleic acid sequences may encode a ribozyme or antisense RNA specificfor the target mRNA. Ribozymes of the hammerhead class are the smallestknown, and lend themselves both to in vitro production and delivery tocells (summarized by Sullivan, 1994, J. Invest. Dermatol., 103: 85S-98S;Usman et al., 1996, Curr. Opin. Struct. Biol., 6: 527-533).

Therapeutic Compositions According to the Invention

Modulators of FANCD2-containing foci formation may be useful therapeuticagents. For example, enhancers of FANCD2-containing foci formation,whether in the presence or absence of genotoxic agents, may be usefulfor providing protection of a subject against genotoxic agents. If acompound which has been identified in a screen of the present inventionor through other means to enhance formation of FANCD2-containing fociand is not itself a genotoxic agent, then such a compound may be auseful as a protective agent. Desferrioxamine (DFO)[1-Amino-6,17-dihydroxy-7,10,18,21-tetraoxo-27-(n-acetylhydroxylamino)-6,11,17,22-tetraazaheptaeicosane;CAS Registry No: 70-51-9] was identified as an agonist of the FA/BRCApathway. DFO is a potent activator of FANCD2 monoubiquitination and fociassembly. DFO is a known chelator of iron, and it is believed todecrease intracellular oxygen radicals (Breuer et al., (2001) Blood, 97,792-798). The identification of DFO as an agonist of the FA/BRCA pathwayhas important implications. Although DFO is a safe drug, in clinicaluse, it may also serve to prime the FA/BRCA DNA repair pathway. As such,it could be a useful and safe protective agent against genotoxic agents.At present there are few known protective agents. For instance,amifostine is approved for radiation protection (Choi, (2003) SeminOncol. 30, 10-17). Thus a therapeutic amount DFO could be administeredto a subject in order to provide protection of the subject fromgenotoxic agents. There are many obvious uses of such an agent whichenhances FANCD2-containing foci formation without itself havinggenotoxic effects, such as during radiation exposure in warfare,following a radioactive spill, or during space travel to Mars.

Likewise, a modulator which reduces the formation of FANCD2-containingfoci may have many uses. An obvious use of an agent which inhibits theFA/BRCA DNA repair pathway would be for its use as a chemosensitizerduring chemotherapy. It is known in the art that any tumors developresistance to chemotherapy. A combination therapy comprising ananticancer therapeutic agent and a chemosensitizer may greatly reducethe resistance of these tumors towards the anticancer agent. Even incases where resistance is not shown to be a problem, it may beadvantageous to administer such a combination therapy if theadministered dose of the anticancer agent can be reduced or if such atreatment diminishes the overall side effects of the anticancer agent.Using the screening methods described in the present invention,Wortmannin and Trichostatin A were identified as inhibitors of theFANCD2-containing foci formation. These compounds, therefore, may beuseful as chemosensitizers.

Pharmaceutical Compositions Comprising Modulators of FANCD2-Containingfoci Formation

In another embodiment, the invention relates to a pharmaceuticalcomposition comprising a, as described in the preceding section, and apharmaceutically acceptable carrier, as described below. Thepharmaceutical composition comprising the modulator of FANCD2-containingfoci formation is useful for treating a variety of diseases anddisorders including cancer, and may be useful as protective agentsagainst genotoxic agents.

The compounds of the present invention, or pharmaceutically acceptablesalts thereof, can be formulated for oral, intravenous, intramuscular,subcutaneous, topical or parenteral administration for the therapeuticor prophylactic treatment of diseases. For oral or parentaladministration, compounds of the present invention can be mixed withconventional pharmaceutical carriers and excipients and used in the formof tablets, capsules, elixirs, suspensions, syrups, wafers and the like.The compositions comprising a compound of this present invention willcontain from about 0.1% to about 99.9%, about 1% to about 98%, about 5%to about 95%, about 10% to about 80% or about 15% to about 60% by weightof the active compound.

The pharmaceutical preparations disclosed herein are prepared inaccordance with standard procedures and are administered at dosages thatare selected to reduce, prevent, or eliminate cancer, or to provide aprotective effect against genotoxic agents such as ionizing radiation.(See, e.g., Remington's Pharmaceutical Sciences, Mack PublishingCompany, Easton, Pa.; and Goodman and Gilman, Pharmaceutical Basis ofTherapeutics, Pergamon Press, New York, N.Y., the contents of which areincorporated herein by reference, for a general description of themethods for administering various antimicrobial agents for humantherapy). The compositions of the present invention can be deliveredusing controlled (e.g., capsules) or sustained release delivery systems(e.g., bioerodable matrices). Exemplary delayed release delivery systemsfor drug delivery that are suitable for administration of thecompositions of the invention are described in U.S. Pat. No. 4,452,775(issued to Kent), U.S. Pat. No. 5,239,660 (issued to Leonard), U.S. Pat.No. 3,854,480 (issued to Zaffaroni).

The pharmaceutically acceptable compositions of the present inventioncomprise one or more compounds of the present invention in associationwith one or more non-toxic, pharmaceutically acceptable carriers and/ordiluents and/or adjuvants and/or excipients, collectively referred toherein as “carrier” materials, and if desired other active ingredients.The compositions may contain common carriers and excipients, such ascorn starch or gelatin, lactose, sucrose, microcrystalline cellulose,kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid.The compositions may contain crosarmellose sodium, microcrystallinecellulose, sodium starch glycolate and alginic acid.

Tablet binders that can be included are acacia, methylcellulose, sodiumcarboxymethylcellulose, polyvinylpyrrolidone (Providone), hydroxypropylmethylcellulose, sucrose, starch and ethylcellulose.

Lubricants that can be used include magnesium stearate or other metallicstearates, stearic acid, silicon fluid, talc, waxes, oils and colloidalsilica.

Flavoring agents such as peppermint, oil of wintergreen, cherryflavoring or the like can also be used. It may also be desirable to adda coloring agent to make the dosage form more aesthetic in appearance orto help identify the product comprising a compound of the presentinvention.

For oral use, solid formulations such as tablets and capsules areparticularly useful. Sustained released or enterically coatedpreparations may also be devised. For pediatric and geriatricapplications, suspension, syrups and chewable tablets are especiallysuitable. For oral administration, the pharmaceutical compositions arein the form of, for example, a tablet, capsule, suspension or liquid.The pharmaceutical composition is preferably made in the form of adosage unit containing a therapeutically-effective amount of the activeingredient. Examples of such dosage units are tablets and capsules. Fortherapeutic purposes, the tablets and capsules which can contain, inaddition to the active ingredient, conventional carriers such as bindingagents, for example, acacia gum, gelatin, polyvinylpyrrolidone,sorbitol, or tragacanth; fillers, for example, calcium phosphate,glycine, lactose, maize-starch, sorbitol, or sucrose; lubricants, forexample, magnesium stearate, polyethylene glycol, silica or talc:disintegrants, for example, potato starch, flavoring or coloring agents,or acceptable wetting agents. Oral liquid preparations generally are inthe form of aqueous or oily solutions, suspensions, emulsions, syrups orelixirs and may contain conventional additives such as suspendingagents, emulsifying agents, non-aqueous agents, preservatives, coloringagents and flavoring agents. Examples of additives for liquidpreparations include acacia, almond oil, ethyl alcohol, fractionatedcoconut oil, gelatin, glucose syrup, glycerin, hydrogenated edible fats,lecithin, methyl cellulose, methyl or propyl para-hydroxybenzoate,propylene glycol, sorbitol, or sorbic acid.

For intravenous (iv) use, compounds of the present invention can bedissolved or suspended in any of the commonly used intravenous fluidsand administered by infusion. Intravenous fluids include, withoutlimitation, physiological saline or Ringer's solution.

Formulations for parental administration can be in the form of aqueousor non-aqueous isotonic sterile injection solutions or suspensions.These solutions or suspensions can be prepared from sterile powders orgranules having one or more of the carriers mentioned for use in theformulations for oral administration. The compounds can be dissolved inpolyethylene glycol, propylene glycol, ethanol, corn oil, benzylalcohol, sodium chloride, and/or various buffers.

For intramuscular preparations, a sterile formulation of compounds ofthe present invention or suitable soluble salts forming the compound,can be dissolved and administered in a pharmaceutical diluent such asWater-for-Injection (WFI), physiological saline or 5% glucose. Asuitable insoluble form of the compound may be prepared and administeredas a suspension in an aqueous base or a pharmaceutically acceptable oilbase, e.g. an ester of a long chain fatty acid such as ethyl oleate.

For topical use the compounds of present invention can also be preparedin suitable forms to be applied to the skin, or mucus membranes of thenose and throat, and can take the form of creams, ointments, liquidsprays or inhalants, lozenges, or throat paints. Such topicalformulations further can include chemical compounds such asdimethylsulfoxide (DMSO) to facilitate surface penetration of the activeingredient.

For application to the eyes or ears, the compounds of the presentinvention can be presented in liquid or semi-liquid form formulated inhydrophobic or hydrophilic bases as ointments, creams, lotions, paintsor powders.

For rectal administration the compounds of the present invention can beadministered in the form of suppositories admixed with conventionalcarriers such as cocoa butter, wax or other glyceride.

Alternatively, the compound of the present invention can be in powderform for reconstitution in the appropriate pharmaceutically acceptablecarrier at the time of delivery. In another embodiment, the unit dosageform of the compound can be a solution of the compound or a salt thereofin a suitable diluent in sterile, hermetically sealed ampoules.

The amount of the compound of the present invention in a unit dosagecomprises a therapeutically-effective amount of at least one activecompound of the present invention which may vary depending on therecipient subject, route and frequency of administration. A recipientsubject refers to a plant, a cell culture or an animal such as an ovineor a mammal including a human.

According to this aspect of the present invention, the novelcompositions disclosed herein are placed in a pharmaceuticallyacceptable carrier and are delivered to a recipient subject (including ahuman subject) in accordance with known methods of drug delivery. Ingeneral, the methods of the invention for delivering the compositions ofthe invention in vivo utilize art-recognized protocols for deliveringthe agent with the only substantial procedural modification being thesubstitution of the compounds of the present invention for the drugs inthe art-recognized protocols.

The compounds of the present invention provide a method for treatingpre-cancerous or cancerous conditions, or for use as a protective agentagainst genotoxic agents. As used herein, the term “unit dosage” refersto a quantity of a therapeutically effective amount of a compound of thepresent invention that elicits a desired therapeutic response. The term“treating” is defined as administering, to a subject, a therapeuticallyeffective amount of at least one compound of the present invention, bothto prevent the occurrence of a pre-cancer or cancer condition, or tocontrol or eliminate pre-cancer or cancer condition. The term “desiredtherapeutic response” refers to treating a recipient subject with acompound of the present invention such that a pre-cancer or cancercondition is reversed, arrested or prevented in a recipient subject.

The compounds of the present invention can be administered as a singledaily dose or in multiple doses per day. The treatment regime mayrequire administration over extended periods of time, e.g., for severaldays or for from two to four weeks. The amount per administered dose orthe total amount administered will depend on such factors as the natureand severity of the disease condition, the age and general health of therecipient subject, the tolerance of the recipient subject to thecompound and the type of cancer, the sensitivity of the cancer totherapeutic agents, and, if used in combination with other therapeuticagent(s), the dose and type of therapeutic agent(s) used.

A compound according to this invention may also be administered in thediet or feed of a patient or animal. The diet for animals can be normalfoodstuffs to which the compound can be added or it can be added to apremix.

The compounds of the present invention may be taken in combination,together or separately with any known clinically approved agent to treata recipient subject in need of such treatment.

Kits According to the Invention

For convenience, the conventional reagents for immunohistochemicalanalysis or immunofluorescent microscopy, or other diagnostic assaysaccording to this invention are provided in the form of kits. Such kitsare useful for determining and enumerating the absence or presence ofFANCD2-containing foci in samples from a living subject. Thus, such akit will be useful in conducting the diagnostic assays in determining ifa subject has recently been exposed to a genotoxic agent, the degree ofexposure of the subject to a genotoxic agent, and the sensitivity of asubject to genotoxic agents. Such a diagnostic kit comprises a FANCD2ligand (e.g., an antibody capable of binding to FANCD2) or amonoubiquitinated FANCD2 ligand of this invention. The kits may alsoinclude instructions for performing the assay, microscopic slides forfixing the tissue or cells, fixatives, suitable stains, various diluentsand buffers, labeled conjugates for the detection of specifically boundcompositions and other signal-generating reagents, such as fluorescentcompounds and dyes, enzyme substrates, cofactors and chromogens.Additional components may include indicator charts for fluorescent orcolorimetric comparisons, disposable gloves, decontaminationinstructions, applicator sticks or containers, and a sample preparatorcup. Such kits provide a convenient, efficient way for a clinicallaboratory to diagnose the presence or absence of DNA damage in a cellor tissue according to this invention. Kits according to the inventioninclude appropriate packaging means, for example test tubes, tissueculture plates and multiwell plates. Ligands can be provided in asolution or in a lyophilized form.

The present invention is described by reference to the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below were utilized.All references cited herein are incorporated by reference.

EXAMPLES Example 1 Development of an Antibody that SpecificallyRecognizes the Monoubiquitinated Isoform of FANCD2

Antibodies to FANCD2 (monoclonal and polyclonal) were raised against theamino terminal region of FANCD2. These antibodies recognize bothisoforms of FANCD2 (FANCD2-S and FANCD2-L). To make an anti-FANCD2-Lantibody, we have generated a specific antigen (FIG. 10), containing aregion of FANCD2 linked covalently to the carboxyl terminus of ubiquitin(Ub). We injected mice with this antigen, and raised monoclonalantibodies (FIG. 11). These monoclonal antibodies will now be analyzedfor their specific diagnostic value, as described above.

Example 2 Clinical Protocol for Taking Samples

Blood is drawn 24 to 48 hours after the alleged exposure, since, basedon in vitro data, this is the peak time of foci formation. Again,different antisera, (say for FANCD2, BRCA1, and Histone 2AX) may differsignificantly in the actual time of peak foci and in the duration offoci in vivo. Peripheral blood lymphocytes (PBLs) are isolated using astandard ficoll gradient. Cells are then stained with specific antiserato Histone 2AX, BRCA1, and FANCD2, and the percentage of cells withIRIFs and the number of IRIFs per cell are measured. Using a standardcurve based on the amount of time which has elapsed since allegedradiation exposure, the likely dose of exposure to genotoxic agents isdetermined. Based on this dose, genotoxic protective agents areadministered if needed.

I. Clinical Protocol

Oncology patients are enrolled who are receiving radiation or genotoxicchemotherapeutic agents for their tumor (head and neck squamous cellcarcinoma patients, for example). Following radiation exposure,peripheral blood samples are drawn at different times after exposure(i.e., 1 hour, 3 hours, 8 hours, and 24 hours post exposure) and thenumber of foci in the peripheral blood lymphocytes are measured. In thecase of samples from patients receiving radiation treatment, while theradiation field will only include the tumor region, blood (andlymphocytes) passing through this field will also receive a radiationexposure. Thus, a peripheral blood sample obtained from the patient willsample at least a fraction of the circulating lymphocytes which wereirradiated in this field. A standard curve is generated correlatingexposure (y-axis) with number of foci x-axis). Subsequently, a highthroughput, automated instrument for measuring the radiation exposure ofmany individual blood samples is developed. This instrument isanticipated to work in the same principle as that used in previousassays to determine the exposure of a biological sample to radiation orgenotoxic agent, in that it comprises contacting the biological samplewith antibodies against proteins found in foci (FANCD2, Histone 2AX,BRCA1) and examining samples for foci formation using high-throughputautomated microscopy.

Example 3 Dose-Dependent Generation of FANCD2 Monoubiquitination

We have previously shown that DNA damage activates FANCD2monoubiquitination and that FANCD2 monoubiquitination correlates withFANCD2 nuclear foci formation (Garcia-Higuera et al., Mol Cell7:249-262, 2001). As shown in FIG. 13, several kinds of genotoxicstresses (MMC, IR, ultraviolet light) can activate FANCD2monoubiquitination (panel A, B, C) or FANCD2 foci formation (panel D).Interestingly, there was a time-course dependent and dose-dependentactivation of FANCD2. In this analysis, FANCD2 foci were observed only 4hours after IR and were observed after 5 Gy of IR (panel D). Morerefined studies (FIG. 12), also performed in HeLa cells, indicate adose-dependent increase in FANCD2 monoubiquitination, even in the 0-5 Gyradiation range (i.e., low radiation exposure), further suggesting thatFANCD2 foci formation may be a very sensitive indicator of radiationexposure. Preliminary studies indicate that irradiation of primaryperipheral blood lymphocytes from normal adult human controls, in vitro,results in similar time-course and dose-dependent generation of FANCD2nuclear foci (data not shown).

Example 4 Screening (Prescreen Using Chemicals)

I) High throughout assay for an inhibitor (or agonist) of the FanconiAnemia/BRCA pathway. An assay has been developed for the assembly ofFANCD2 foci, a critical downstream event in the FA/BRCA pathway. Forthis purpose a fusion cDNA (FIG. 1) which encodes the GFP (greenfluorescence protein), fused at the amino terminus of the full lengthFANCD2 protein, has been generated.

Initially, this cDNA was transfected into PD20 (FA-D2 fibroblasts),which express no endogenous FANCD2 protein (FIG. 2) The GFP-FANCD2protein is larger than FANCD2 (as predicted) and undergoes DNAdamage-inducible monoubiquitination (lanes 6-9). When the GFP-FANCD2protein was expressed in an FA-A (Fanconi Anemia subtype A) Fibroblastline, it was not monoubiquitinated, even when the cells were exposed toIonizing Radiation (FIG. 2, lanes 11-14). Taken together, these resultsindicate that GFP-FANCD2 behaves similarly to the wild-type (untagged)FANCD2 protein.

Next it was determined whether GFP-FANCD2 can correct the MMChypersensitivity of PD2OF cells (FIG. 3). Indeed, expression ofGFP-FANCD2, like wild-type FANCD2, restores normal MMC resistance.

The PD2OF cells, expressing GFP-FANCD2 were plated and individualsubclones isolated. One subclone (clone 7) expressed GFP-FANCD2 proteindiffusely in its nucleus (FIG. 4). Following cellular exposure to IR,those cells formed bright, green foci in the nucleus. Thus, these cellsare an ideal tool for high-throughput screening of antagonists andagonists of the FA/BRCA pathway.

II. Use of GFP-FANCD2 Expressing Fibroblasts to Identify Bioactive SmallMolecule Regulators of the FA/BRCA Pathway.

A screening test was established in a core facility at Harvard MedicalSchool called the ICCB (Institute for Chemistry and Chemical Biology).The general principle of this screening assay is shown in FIG. 5.

A small molecule transfer robot delivers chemical compounds to clone 7fibroblasts, plated in 384-cell tissue culture plates. Severalcommercial libraries are available, comprising over 200,000 smallmolecules. Inhibitors of the FA/BRCA pathway are expected to blockGFP-FANCD2 foci formation; agonists of the pathway will promote fociformation.

The specific details of the assay are described in FIG. 6. As shown, theplated cells are preincubated with compounds (approximately 40micromolar concentrations for twelve hours) before the stimulation withIonizing Radiation. An important feature of the protocol is the use ofsecondary screens (FIG. 6, Point #8). Any compound initially found toinhibit or activate (i.e., synergize with) FANCD2 foci formation issubsequently screened in lower dose ranges (1-20 micromolar range) andis screened by two assays: (1) formation of FANCD2 foci and (2)activation of FANCD2 monoubiquitination (Western blot screen). Anycompound which passes this secondary screen will then be examined forits ability to chemosensitize HeLa cells to the cytotoxic effects ofcisplatin. (Taniguchi et al., (2003) Nat. Med 9:568-574).

III. Identification of Specific Compounds which Inhibit or Activate theFANCD2 Pathway.

Photomicrographs of individual cells from the screening assay are shownin FIG. 7. Initially, approximately 1000 known bioactive compounds werescreened. An agent which reduces by at least 10%, for example 10%, 20%,30%, 50%, 75%, or up to 100%, the number and size of foci formed uponexposure to genotoxic agents such as ionizing radiation (IR) isindicative of an agent which inhibits formation of FANCD2-containingfoci. In another screen, an agent which, in the absence of exposure togenotoxic agent, causes an at least 10%, for example 10%, 20%, 30%, 50%,75%, 100%, 200% or 500%, increase in formation of FANCD2-containing focirelative to unexposed control cells is indicative of an agent whichactivates formation of FANCD2-containing foci. The vast majority ofthese compounds had no effect on the ability of Clone 7 cells to formGFP-FANCD2 foci after exposure IR. At least three relevant compoundsemerged from the initial screen, two functioning as inhibitors of thepathway, and one functioning as an activator of the pathway.

A) Inhibitors of the FA/BRCA Pathway.

One inhibitor of the FA/BRCA pathway was the compound wortmannin.Wortmannin is a known inhibitor of the DNA damage response kinases, ATMand ATR. ATR kinase has been shown to be an upstream component of theFA/BRCA pathway, functioning as part of the molecular sensor apparatusof the pathway (see FIG. 8, below).

More selective libraries of compounds, enriched with novel kinaseinhibitors are screened to determine if any of these compounds actspecifically as inhibitors of the FA/BRCA pathway.

Another inhibitor of the FA/BRCA pathway identified was Trichostatin-A.Trichostatin-A is a known inhibitor of a broad class of enzymes known asHDACs (Histone Deacetylases). There are at least eight different HDACenzymes in human cells. Importantly, HDAC inhibitors, such asTrichostatin-A (Beppu et al., (1990) J Biol. Chem. 265, 17174-9) andSAHA (Richon et al., Proc Natl Acad Sci USA. (1998) 95, 3003-7), havepotent anti-tumor activity in vitro and in vivo, and this class of drugsis currently under intense investigation as a new class of humananti-neoplastic agents.

The identification (and confirmation) of Trichostatin-A as an inhibitorof the FA/BRCA pathway has important implications. For instance:

-   1) Now, other HDAC inhibitors, some of which are in clinical trials    in cancer patients, can be tested directly for their ability to    inhibit the FA/BRCA pathway. In fact, FA/BRCA pathway in clone 7    cells may provide a useful biomarker for the effectiveness of new    HDAC inhibitors.-   2) While HDAC inhibitors have broad effects on cellular function    (affecting DNA repair, transcription, and mitogenesis), in fact the    disruption of the FA/BRCA pathway may be the relevant “readout” in    assessing the potency of a given HDAC inhibitor derivative compound.-   3) HDAC inhibitors may in fact function as radiosensitizers and    chemosensitizers of cancer cells, by inhibiting DNA repair through    the FA/BRCA pathway. Accordingly, HDAC inhibitors may be delivered    most effectively in combination with radiation or cytotoxic DNA    damaging drugs (i.e., cisplatin).    B) Agonists of the FA/BRCA Pathway.

Desferrioxamine (DFO) was identified as an agonist of the FA/BRCApathway. DFO is a potent activator of FANCD2 monoubiquitination and fociassembly. DFO is a known chelator of iron, and it is believed todecrease intracellular oxygen radicals, but its role in activating theFANCD2 monoubiquitination and foci formation has not been previouslydemonstrated.

Example 5 Determination of the Molecular Sensor Apparatus of the FA/BRCAPathway

Wortmannin, a known inhibitor of ATM and ATR kinases, was shown to blockFANCD2 monoubiquitination and foci formation. Accordingly, this resultsuggested to us that ATM (or ATR) may function upstream in the FA/BRCApathway. To test this hypothesis, an siRNA strategy was used (FIG. 8, A,B). Inhibitory RNA molecules, specific for ATR, CHK1, and RPA1 (RPA1 isa known activator of ATR; CHK1 is a known substrate of ATR.)Interestingly, transient transfection of HeLa cells with these siRNAmolecules (1) reduced MMC-inducible and IR-inducible monoubiquitinationof FANCD2 (FIG. 8), (2) reduced FANCD2 foci formation (not shown) andsensitized the HeLa cells to MMC (not shown). An important feature ofthis assay (FIG. 8) is the calculation of the “L/S ratio.” This ratio iscalculated as the density of the FANCD2-L band divided by the density ofthe FANCD2-S band. These band intensities are determined directly fromthe autoradiograph. For instance (FIG. 8A), MMC activation results in anincrease of the L/S ratio to 1.17; knockout of ATR, with siRNA, reducesthe L/S ratio to 0.46 (unitless value).

Based on these important observations, it is clear that theATR/RPA1/CHK1 network of proteins works upstream in the FA/BRCA pathway(FIG. 9). Disruption of any of the steps upstream in this pathway,through pharmacological manipulation, can potentially radiosensitize orchemosensitize cancer cells to other conventional antineoplasticreagents.

Uses

The invention is useful in the detection of exposure of a living subjectto genotoxic agents such as ionizing radiation. In addition, theinvention is useful in determining the sensitivity of a living subjectto genotoxic agents prior to exposure of such agents, for example forradiation therapy, and is useful in providing a more individualizedtherapy with reduced risk of overexposure of sensitive patients. Theinvention is also of use in identifying agents which alter the degree offoci formation. These agents could be useful as protective agentsagainst DNA damage induced by genotoxic agents, or alternatively couldbe useful as chemosensitizing agents to be used in combination withchemotherapeutic drugs.

Other Embodiments

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

REFERENCES FOR THIS SECTION

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1. A method of detecting exposure to a genotoxic agent in a livesubject, comprising: collecting a sample from said subject; anddetecting the presence of FANCD2-containing foci in said sample, whereinthe presence of foci is indicative of exposure to a genotoxic agent. 2.The method of claim 1, wherein said subject is human.
 3. The method ofclaim 1, wherein said sample is selected from a group consisting ofperipheral blood, saliva, urine, a cell scraping, exudate, a buccalsample, sputum, and cervical scraping.
 4. The method of claim 1, whereinsaid sample is peripheral blood.
 5. The method of claim 1, furthercomprising a control sample.
 6. The method of claim 5, wherein thedegree of foci formation relative to a control sample is indicative ofthe degree of exposure to a genotoxic agent.
 7. The method of claim 1,further comprising contacting a sample with a ligand which binds tohuman FANCD2 of SEQ ID NO:1, said ligand associated with a label whichprovides a detectable signal.
 8. The method of claim 7, wherein saidligand is an antibody.
 9. The method of claim 7, wherein said label isattached to said antibody.
 10. The method of claim 7, wherein said labelis attached to a second ligand, which binds to the first ligand.
 11. Themethod of claim 10, wherein said second ligand is an antibody.
 12. Themethod of claim 7, wherein said label is selected from the groupconsisting of colorimetric, chemiluminescent, fluorescent,electrochemical labels and combinations thereof.
 13. The method of claim12, wherein said label is a fluorescent dye.
 14. The method of claim 1,wherein said detecting comprises fluorescence microscopy.
 15. A methodof testing a patient's sensitivity to a genotoxic agent comprising:exposing a patient to a low dose of genotoxic agent; and, detecting thepresence of FANCD2-containing foci relative to a control sample, whereinthe presence of foci formation relative to a control sample isindicative of a difference in sensitivity of said patient to genotoxicagent.
 16. The method of claim 15, wherein said sample is selected froma group consisting of peripheral blood, saliva, urine, a cell scraping,exudate, a buccal sample, sputum, and cervical scraping.
 17. The methodof claim 15, wherein said sample is peripheral blood.
 18. The method ofclaim 15, wherein the degree of foci formation relative to a controlsample is indicative of the sensitivity of said patient to genotoxicagent.
 19. The method of claim 15, further comprising contacting asample with a ligand which binds to human FANCD2 of SEQ ID NO:1, saidligand associated with a label which provides a detectable signal. 20.The method of claim 19, wherein said ligand is an antibody.
 21. Themethod of claim 19, wherein said label is attached to said antibody. 22.The method of claim 19, wherein said label is attached to a secondligand, which binds to the first ligand.
 23. The method of claim 22,wherein said second ligand is an antibody.
 24. The method of claim 19,wherein said label is selected from the group consisting ofcalorimetric, chemiluminescent, fluorescent, electrochemical labels andcombinations thereof.
 25. The method of claim 24, wherein said label isa fluorescent dye.
 26. The method of claim 15, wherein said detectingcomprises fluorescence microscopy.
 27. A method of determining the levelof DNA damage caused by exposure of subject to a genotoxic agent,comprising: collecting a sample from said patient following saidexposure; and detecting the presence of FANCD2-containing foci relativeto a control sample, wherein a difference in foci formation relative tosaid control sample is indicative of a difference in DNA damage inresponse to said exposure, and wherein the degree of foci formationrelative to a control sample is indicative of a different extent of DNAdamage in response to said exposure.
 28. The method of claim 27, whereinsaid sample is selected from a group consisting of peripheral blood,saliva, urine, a cell scraping, exudate, a buccal sample, sputum, andcervical scraping.
 29. The method of claim 27, wherein said sample isperipheral blood.
 30. The method of claim 27, further comprisingcontacting a sample with a ligand which binds to human FANCD2 of SEQ IDNO:1, said ligand associated with a label which provides a detectablesignal.
 31. The method of claim 30, wherein said ligand is an antibody.32. The method of claim 30, wherein said label is attached to saidantibody.
 33. The method of claim 30, wherein said label is attached toa second ligand, which binds to the first ligand.
 34. The method ofclaim 33, wherein said second ligand is an antibody.
 35. The method ofclaim 30, wherein said label is selected from the group consisting ofchemiluminescent compounds, fluorescent dyes, chromogenic dyes,electrochemical tags and combinations thereof.
 36. The method of claim35, wherein said label is a fluorescent dye.
 37. The method of claim 36,wherein said detecting comprises fluorescence microscopy.
 38. Anisolated polynucleotide comprising a DNA sequence encoding the humanFACD2 protein of SEQ ID NO:1 fused in frame with a DNA sequence encodinga fluorescent protein.
 39. The isolated polynucleotide of claim 38,further comprising an expression control sequence operatively linked tosaid sequence encoding said FANCD2 protein fused in frame with a DNAsequence encoding a fluorescent protein.
 40. The genetic construct ofclaim 38, wherein the fluorescent protein is selected from the groupincluding: GFP, YFP, CFP, eGFP, eYFP, eCFP, RFP.
 41. A protein encodedby the isolated polynucleotide of claim
 38. 42. A cell expressing theisolated polynucleotide of claim
 38. 43. An isolated polynucleotidecomprising a DNA sequence encoding a protein that binds with FANCD2 uponfoci formation, fused in frame with a DNA sequence encoding a secondfluorophore.
 44. The isolated polynucleotide of claim 43, furthercomprising an expression control sequence operatively linked to DNAsequences encoding said FANCD2 protein fused in frame to a DNA sequenceencoding a fluorescent protein.
 45. The isolated polynucleotide of claim43, wherein the fluorescent protein is selected from the groupincluding: GFP, YFP, CFP, eGFP, eYFP, eCFP, RFP.
 46. The isolatedpolynucleotide of claim 43, further comprising a protein linkersequence.
 47. A protein encoded by the isolated polynucleotide of claim43.
 48. A cell expressing the isolated polynucleotide of claim
 43. 49. Acell expressing the isolated polynucleotide of claim 38 and
 43. 50. Amethod of screening test agents which modulate formation ofFANCD2-containing foci, said method comprising: contacting a biologicalsample with test compound; and detecting the presence of FANCD2 inFANCD2-containing foci relative to a control sample, wherein adifference in the degree of foci formation relative to a control sampleis indicative of an agent active in foci formation.
 51. The method ofclaim 50, further comprising exposing biological sample to a genotoxicagent.
 52. The method of claim 50, further comprising contacting saidbiological sample with a ligand which binds to FANCD2, said ligandassociated with a label which provides a detectable signal.
 53. Themethod of claim 52, wherein said ligand is an antibody.
 54. The methodof claim 52, wherein said label is attached to a second ligand, whichbinds to the first ligand.
 55. The method of claim 52, wherein saidsecond ligand is an antibody.
 56. The method of claim 52, wherein saidlabel is selected from the group consisting of chemiluminescentcompounds, fluorescent dyes, chromogenic dyes, electrochemical tags andcombinations thereof.
 57. The method of claim 56, wherein said label isa fluorescent dye.
 58. The method of claim 50, wherein said biologicalsample is the cell of claim
 42. 59. The method of claim 50, wherein saidbiological sample is the cell of claim
 48. 60. The method of claim 50,wherein said biological sample is the cell of claim
 49. 61. The methodof claim 50, wherein said detecting comprises fluorescence microscopy.62. The method of claim 50, wherein said detecting comprises measuringfluorescence resonance energy transfer.
 63. An antibody that binds to amonoubiquitinated form of FANCD2 polypeptide and ubiquitin.
 64. Theantibody of claim 63, wherein said antibody is polyclonal.
 65. Theantibody of claim 63, wherein said antibody is monoclonal.
 66. A kit fordetecting the presence or absence of FANCD2-containing foci in a samplefrom a live subject comprising the antibody of claim 63, and packagingmaterial.
 67. The kit of claim 66, further comprising fluorescentlylabeled secondary antibody, wherein said secondary antibody binds to thefirst said antibody.
 68. The kit of claim 66, further comprising theisolated polynucleotides of claims 38 and 43.